Aryl-fused spirocyclic compounds

ABSTRACT

The present invention relates to a novel class of aryl-fused spirocyclic compounds. These compounds can inhibit histone deacetylase and are suitable for use in selectively inducing terminal differentiation, and arresting cell growth and/or apoptosis of neoplastic cells, thereby inhibiting proliferation of such cells. Thus, the compounds of the present invention are useful in treating a patient having a tumor characterized by proliferation of neoplastic cells. The compounds of the invention may also be useful in the prevention and treatment of TRX-mediated diseases, such as autoimmune, allergic and inflammatory diseases, and in the prevention and/or treatment of diseases of the central nervous system (CNS), such as neurodegenerative diseases. The present invention further provides pharmaceutical compositions comprising the compounds of the instant invention and safe dosing regimens of these pharmaceutical compositions, which are easy to follow, and which result in a therapeutically effective amount of these compounds in vivo.

PRIORITY CLAIM

This application is a §371 application of PCT/US07/011,540 that wasfiled on May 14, 2007, which claims priority from the U.S. ProvisionalApplication No. 60/801,269, filed on May 18, 2006, now expired.

FIELD OF THE INVENTION

The present invention relates to a novel class of aryl-fused spirocycliccompounds. These compounds can inhibit histone deacetylase and aresuitable for use in selectively inducing terminal differentiation, andarresting cell growth and/or apoptosis of neoplastic cells, therebyinhibiting proliferation of such cells. Thus, the compounds of thepresent invention are useful in treating a patient having a tumorcharacterized by proliferation of neoplastic cells. The compounds of theinvention may also be useful in the prevention and treatment ofTRX-mediated diseases, such as autoimmune, allergic and inflammatorydiseases, and in the prevention and/or treatment of diseases of thecentral nervous system (CNS), such as neurodegenerative diseases.

BACKGROUND OF THE INVENTION

The inhibition of HDACs can repress gene expression, includingexpression of genes related to tumor suppression. Inhibition of histonedeacetylase can lead to the histone deacetylase-mediated transcriptionalrepression of tumor suppressor genes. For example, inhibition of histonedeacetylase can provide a method for treating cancer, hematologicaldisorders, such as hematopoiesis, and genetic related metabolicdisorders. More specifically, transcriptional regulation is a majorevent in cell differentiation, proliferation, and apoptosis. There areseveral lines of evidence that histone acetylation and deacetylation aremechanisms by which transcriptional regulation in a cell is achieved(Grunstein, M., Nature, 389: 349-52 (1997)). These effects are thoughtto occur through changes in the structure of chromatin by altering theaffinity of histone proteins for coiled DNA in the nucleosome. There arefive types of histones that have been identified. Histones H2A, H2B, H3and H4 are found in the nucleosome, and H1 is a linker located betweennucleosomes. Each nucleosome contains two of each histone type withinits core, except for H1, which is present singly in the outer portion ofthe nucleosome structure. It is believed that when the histone proteinsare hypoacetylated, there is a greater affinity of the histone to theDNA phosphate backbone. This affinity causes DNA to be tightly bound tothe histone and renders the DNA inaccessible to transcriptionalregulatory elements and machinery.

The regulation of acetylated states occurs through the balance ofactivity between two enzyme complexes, histone acetyl transferase (HAT)and histone deacetylase (HDAC).

The hypoacetylated state is thought to inhibit transcription ofassociated DNA. This hypoacetylated state is catalyzed by largemultiprotein complexes that include HDAC enzymes. In particular, HDACshave been shown to catalyze the removal of acetyl groups from thechromatin core histones.

It has been shown in several instances that the disruption of HAT orHDAC activity is implicated in the development of a malignant phenotype.For instance, in acute promyelocytic leukemia, the oncoprotein producedby the fusion of PML and RAR alpha appears to suppress specific genetranscription through the recruitment of HDACs (Lin, R. J. et al.,Nature 391:811-14 (1998)). In this manner, the neoplastic cell is unableto complete differentiation and leads to excess proliferation of theleukemic cell line.

U.S. Pat. Nos. 5,369,108, 5,932,616, 5,700,811, 6,087,367 and 6,511,990,the contents of which are hereby incorporated by reference, disclosehydroxamic acid derivatives useful for selectively inducing terminaldifferentiation, cell growth arrest or apoptosis of neoplastic cells. Inaddition to their biological activity as antitumor agents, thesehydroxamic acid derivatives have recently been identified as useful fortreating or preventing a wide variety of thioredoxin (TRX)-mediateddiseases and conditions, such as inflammatory diseases, allergicdiseases, autoimmune diseases, diseases associated with oxidative stressor diseases characterized by cellular hyperproliferation (U.S.application Ser. No. 10/369,094, filed Feb. 15, 2003, the entire contentof which is hereby incorporated by reference). Further, these hydroxamicacid derivatives have been identified as useful for treating diseases ofthe central nervous system (CNS) such as neurodegenerative diseases andfor treating brain cancer (See, U.S. application Ser. No. 10/273,401,filed Oct. 16, 2002, the entire content of which is hereby incorporatedby reference).

In view of the wide variety of applications for compounds containinghydroxamic acid moieties, the development of new inhibitors havingimproved properties, for example, increased potency or increasedbioavailability is highly desirable.

SUMMARY OF THE INVENTION

The present invention relates to a novel class of aryl-fused spirocycliccompounds. These compounds, which can be used to treat cancer, inhibithistone deacetylase and are suitable for use in selectively inducingterminal differentiation, and arresting cell growth and/or apoptosis ofneoplastic cells, thereby inhibiting proliferation of such cells. Thus,the compounds of the present invention are useful in treating a patienthaving a tumor characterized by proliferation of neoplastic cells. Thecompounds of the invention may also be useful in the prevention andtreatment of TRX-mediated diseases, such as autoimmune, allergic andinflammatory diseases, and in the prevention and/or treatment ofdiseases of the central nervous system (CNS), such as neurodegenerativediseases. The present invention further provides pharmaceuticalcompositions comprising the compounds of the instant invention, andsafe, dosing regimens of these pharmaceutical compositions, which areeasy to follow, and which result in a therapeutically effective amountof these compounds in vivo.

The present invention relates to compounds represented by the followingstructural formula and pharmaceutically acceptable salts, solvates andhydrates thereof, as detailed herein.

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel class of aryl-fused spirocycliccompounds. The compounds of the instant invention can inhibit histonedeacetylase and are suitable for use in selectively inducing terminaldifferentiation, and arresting cell growth and/or apoptosis ofneoplastic cells, thereby inhibiting proliferation of such cells. Thus,the compounds of the present invention are useful in treating cancer ina subject. The compounds of the invention may also be useful in theprevention and treatment of TRX-mediated diseases, such as autoimmune,allergic and inflammatory diseases, and in the prevention and/ortreatment of diseases of the central nervous system (CNS), such asneurodegenerative diseases.

The present invention relates to compounds represented by the followingstructural formula:

wherein X is

A, B, D, E, G, J, K, M and P are independently selected from CR¹ ₂,NR^(1a), C(O) and O;R is NH₂ or OH;

is aryl or heteroaryl, optionally substituted with from 1 to 3substituents selected from R⁷;

is aryl or heteroaryl;each R¹ is independently

-   -   1) hydrogen,    -   2) C₁-C₆ alkyl,    -   3) (CR⁶ ₂)_(n)R¹⁰,    -   4) (CR⁶ ₂)_(n)C(O)R⁴,    -   5) (CR⁶ ₂)_(n)C(O)OR⁴,    -   6) (CR⁶ ₂)_(n)C(O)NR⁵ ₂,    -   7) (CR⁶ ₂)_(n)S(O)₂R⁴,    -   8) (CR⁶ ₂)_(n)OR⁴,    -   9) (CR⁶ ₂)_(n)NR⁵(SO₂)R⁴,    -   10) (CR⁶ ₂)_(n)NR⁵ ₂    -   11)(CR⁶ ₂)_(n)NR⁵C(O)OR⁴,    -   12) halo, or    -   13) aryl;        each R^(1a) is independently    -   1) hydrogen,    -   2) C₁-C₆ alkyl,    -   3) (CR⁶ ₂)_(n)R¹⁰,    -   4) (CR⁶ ₂)_(n)C(O)R⁴,    -   5) (CR⁶ ₂)_(n)C(O)OR⁴,    -   6) (CR⁶ ₂)_(n)C(O)NR⁵ ₂, or    -   7) (CR⁶ ₂)_(n)S(O)₂R⁴;        L¹ is a bond, —(CR¹¹)_(r)—, —C(O)NR⁵—, —NR⁵C(O)—, or —C(O)—;    -   wherein r is 1, 2 or 3;        R³ is    -   1) hydrogen,    -   2) unsubstituted or substituted C₁-C₆ alkyl,    -   3) unsubstituted or substituted aryl,    -   4) unsubstituted or substituted heteroaryl    -   5) halo,    -   6) CN,    -   7) amide,    -   8) carboxyl,    -   9) C₁-C₇ alkoxy,    -   10) C₁-C₇ haloalkyl,    -   11) C₁-C₇ haloalkyloxy,    -   12) C₁-C₇ hydroxyalkyl,    -   13) C₁-C₇ alkenyl,    -   14) C₁-C₇ alkynyl,    -   15) C₁-C₇ alkyl-C(═O)O—,    -   16) C₁-C₇ alkyl-C(═O)—,    -   17) hydroxyalkoxy,    -   18) —NHSO₂,    -   19) —SO₂NH,    -   20) C₁-C₇ alkyl-NHSO₂—,    -   21) C₁-C₇ alkyl-SO₂NH—,    -   22) C₁-C₇ alkylsulfonyl,    -   23) C₁-C₇ alkylamino,    -   24) di(C₁-C₇)alkylamino, or    -   25) L²-R¹²;        each R⁴ is independently    -   1) H,    -   2) C₁-C₆ alkyl,    -   3) aryl, or    -   4) heterocyclyl,    -   wherein alkyl, aryl or heterocyclyl is optionally substituted        with C₁-C₆ alkyl;        each R⁵ is independently    -   1) hydrogen,    -   2) C₁-C₆ alkyl, or    -   3) aryl,    -   which may be optionally substituted with 1 to 3 substituents        selected from C₁-C₆ alkyl, aryl, heteroaryl or halo;        each R⁶ is independently    -   1) hydrogen,    -   2) C₁-C₆ alkyl,    -   3) aryl,    -   4) OR¹¹,    -   5) halo, or    -   6) NR¹¹;    -   wherein the alkyl or aryl may be optionally substituted with 1        to 3 substituents selected from the group consisting of C₁-C₆        alkyl, aryl, heteroaryl and halo;        each R⁷ is independently hydrogen, OH, NR¹¹ ₂, nitro, CN, amide,        carboxyl, C₁-C₇ alkoxy, C₁-C₇ alkyl, C₁-C₇ haloalkyl, C₁-C₇        haloalkyloxy, C₁-C₇ hydroxyalkyl, C₁-C₇ alkenyl, C₁-C₇        alkyl-C(═O)O—, C₁-C₇ alkyl-C(═O)—, C₁-C₇ alkynyl, halo group,        amide, hydroxyalkoxy, —NR¹¹SO₂, —SO₂NR¹¹ ₂, C₁-C₇alkyl-NR¹¹SO₂—,        C₁-C₇alkyl-SO₂NR¹¹—, C₁-C₇alkylsulfonyl, C₁-C₇ alkylamino or        di(C₁-C₇)alkylamino;        each R¹⁰ is independently    -   1) aryl, and    -   2) heterocyclyl,    -   which may be optionally substituted;        each R¹¹ is independently hydrogen, C₁-C₆ alkyl, or aryl,        wherein said alkyl and aryl groups are optionally substituted;        L² is    -   1) a bond,    -   2) C₁-C₄ alkylene,    -   3) C₁-C₄ alkynyl,    -   4) C₁-C₄ alkenyl,    -   5) —O—,    -   6) —S—,    -   7) —NH—,    -   8) —C(═O)NH—,    -   9) —NHC(═O)—,    -   10) —NHC(═O)NH—,    -   11) —SO₂NH—,    -   12) —NHSO₂—,    -   13) —SO₂—,    -   14) —C(═O)— or    -   15) —C(═O)O—;        R¹² is    -   1) substituted or unsubstituted heteroaryl,    -   2) substituted or unsubstituted heterocyclyl,    -   3) substituted or unsubstituted aryl, and    -   4) substituted or unsubstituted C₃-C₈ cycloalkyl;        n is independently selected from 0, 1, 2, 3 and 4;        q is 1, 2, 3, or 4;        or a stereoisomer or a pharmaceutically acceptable salt thereof.

In an embodiment of the invention, R is NH₂.

In another embodiment of the invention,

is heteroaryl. In a class of the invention,

is pyridinyl.

In another embodiment of the invention,

is aryl. In a class of the invention,

is phenyl.

In another embodiment of the invention, R³ is hydrogen, unsubstituted orsubstituted aryl or unsubstituted or substituted heteroaryl. In a classof the invention, R³ is phenyl or thienyl.

In another embodiment of the invention, L¹ is a bond.

In another embodiment of the invention, nonlimiting examples of Xinclude:

Specific embodiments depicting non-limiting examples of the compounds ofthe instant invention are provided in the experimental sectionhereinbelow.

Specific examples of the compounds of the instant invention include, butare not limited to:

-   N-(2-aminophenyl)-6-(1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl)nicotinamide;-   6-(1-acetyl-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl)-N-(2-aminophenyl)nicotinamide;-   6-(1-acetyl-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl)-N-[2-amino-5-(2-thienyl)phenyl]nicotinamide;-   6-(1-acetyl-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl)-N-[2-amino-5-(3-thienyl)phenyl]nicotinamide;-   N-(2-aminophenyl)-6-{1-[(4-methylphenyl)sulfonyl]-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl}nicotinamide;-   N-(4-aminobiphenyl-3-yl)-6-(2,3-dihydro-1H,1′H-spiro[isoquinoline-4,4′-piperidin]-1′-yl)nicotinamide;-   N-(2-aminophenyl)-6-(1′H-spiro[indeno[2,1-b]pyridine-9,4′-piperidin]-1′-yl)nicotinamide;-   N-(2-aminophenyl)-6-(1,3-diethyl-4,5-dihydro-1H,1′H-spiro[cyclopenta[c]pyrazole-6,4′-piperidin]-1′-yl)nicotinamide;-   N-(2-aminophenyl)-6-(3-oxo-2,3-dihydro-1′H-spiro[isoindole-1,4′-piperidin]-1′-yl)nicotinamide;-   (3R)-1′-(5-{[(2-aminophenyl)amino]carbonyl}pyridin-2-yl)-N-methyl-2,3-dihydrospiro[indene-1,4′-piperidine]-3-carboxamide;-   N-[2-amino-5-(3-thienyl)phenyl]-6-(4-oxo-3,4-dihydro-1′H-spiro[chromene-2,4′-piperidin]-1′-yl)nicotinamide;-   N-(4-aminobiphenyl-3-yl)-6-(1-oxo-1,4-dihydro-1′H-spiro[isochromene-3,4′-piperidin]-1′-yl)nicotinamide;-   ethyl[1′-(5-{[(4-aminobiphenyl-3-yl)amino]carbonyl}pyridin-2-yl)-2,3-dihydrospiro[indene-1,4′-piperidin]-3-yl]carbamate;-   N-4-aminobiphenyl-3-yl)-6-(6-methoxy-1-oxo-4-phenyl-1,4-dihydro-1H′-spiro[isochromene-3,4′-piperidin]-1′-yl)nicotinamide;-   N-(2-aminophenyl)-6-(1-methyl-2-oxo-5-phenyl-1,2-dihydro-1′H-spiro[1,4-benzodiazepine-3,4′-piperidin]-1′-yl)nicotinamide;-   N-(2-aminophenyl)-6-(1-isopropyl-6-methoxy-5-methyl-2-oxo-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl)nicotinamide;-   N-(2-aminophenyl)-6-[5-(benzylamino)-1-isopropyl-2-oxo-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl]nicotinamide;-   N-[2-amino-5-(2-thienyl)phenyl]-6-(1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl)nicotinamide;-   N-[2-amino-5-(2-thienyl)phenyl]-6-(1′H-spiro[1-benzofuran-3,4′-piperidin]-1′-yl)nicotinamide;-   N-[2-amino-5-(2-thienyl)phenyl]-6-[1-(methylsulfonyl)-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl]nicotinamide;-   tert-butyl    1′-(5-{[(2-aminophenyl)amino]carbonyl}pyridin-2-yl)-1H-spiro[isoquinoline-4,4′-piperidine]-2(3H)-carboxylate;-   N-(2-aminophenyl)-6-(2,3-dihydro-1H,1′H-spiro[isoquinoline-4,4′-piperidin]-1′-yl)nicotinamide;-   N-(4-aminobiphenyl-3-yl)-4-(1H-spiro[isoquinoline-4,4′-piperidin]-2(3H)-ylmethyl)benzamide;-   N-(4-aminobiphenyl-3-yl)-4-(2,3-dihydro-1H,1′H-spiro[isoquinoline-4,4′-piperidin]-1′-ylcarbonyl)benzamide;-   N-(4-{[(4-aminobiphenyl-3-yl)amino]carbonyl}phenyl)-2,3-dihydro-1H,1′H-spiro[isoquinoline-4,4′-piperidine]-1′-carboxamide;    or a stereoisomer or a pharmaceutically acceptable salt thereof.    Chemical Definitions

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms. For example, C₁-C₁₀, as in “C₁-C₁₀alkyl” is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 carbons in a linear or branched arrangement. For example, “C₁-C₁₀alkyl” specifically includes methyl, ethyl, n-propyl, i-propyl, n-butyl,t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and so on.The term “cycloalkyl” means a monocyclic saturated aliphatic hydrocarbongroup having the specified number of carbon atoms. The cycloalkyl isoptionally bridged (i.e., forming a bicyclic moiety), for example with amethylene, ethylene or propylene bridge. The bridge may be optionallysubstituted or branched. The cycloalkyl may be fused with an aryl groupsuch as phenyl, and it is understood that the cycloalkyl substituent isattached via the cycloalkyl group. For example, “cycloalkyl” includescyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl,2-ethyl-cyclopentyl, cyclohexyl, and so on. In an embodiment of theinvention the term “cycloalkyl” includes the groups describedimmediately above and further includes monocyclic unsaturated aliphatichydrocarbon groups. For example, “cycloalkyl” as defined in thisembodiment includes cyclopropyl, methyl-cyclopropyl,2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, cyclopentenyl,cyclobutenyl and so on. In an embodiment, if the number of carbon atomsis not specified, “alkyl” refers to C₁-C₁₂ alkyl and in a furtherembodiment, “alkyl” refers to C₁-C₆ alkyl. In an embodiment, if thenumber of carbon atoms is not specified, “cycloalkyl” refers to C₃-C₁₀cycloalkyl and in a further embodiment, “cycloalkyl” refers to C₃-C₇cycloalkyl. In an embodiment, examples of “alkyl” include methyl, ethyl,n-propyl, i-propyl, n-butyl, t-butyl and i-butyl.

The term “alkylene” means a hydrocarbon diradical group having thespecified number of carbon atoms. For example, “alkylene” includes—CH₂—, —CH₂CH₂— and the like. In an embodiment, if the number of carbonatoms is not specified, “alkylene” refers to C₁-C₁₂ alkylene and in afurther embodiment, “alkylene” refers to C₁-C₆ alkylene.

When used in the phrases “alkylaryl”, “alkylcycloalkyl” and“alkylheterocyclyl” the term “alkyl” refers to the alkyl portion of themoiety and does not describe the number of atoms in the aryl andheteroaryl portion of the moiety. In an embodiment, if the number ofcarbon atoms is not specified, “alkyl” of “alkylaryl”, “alkylcycloalkyl”and “alkylheterocyclyl” refers to C₁-C₁₂ alkyl and in a furtherembodiment, the term refers to C₁-C₆ alkyl.

If no number of carbon atoms is specified, the term “alkenyl” refers toa non-aromatic hydrocarbon radical, straight, branched or cyclic,containing from 2 to 10 carbon atoms and at least one carbon to carbondouble bond. Preferably one carbon to carbon double bond is present, andup to four non-aromatic carbon-carbon double bonds may be present. Thus,“C₂-C₆ alkenyl” means an alkenyl radical having from 2 to 6 carbonatoms. Alkenyl groups include ethenyl, propenyl, butenyl,2-methylbutenyl and cyclohexenyl. The straight, branched or cyclicportion of the alkenyl group may contain double bonds and may besubstituted if a substituted alkenyl group is indicated.

The term “alkynyl” refers to a hydrocarbon radical straight, branched orcyclic, containing from 2 to 10 carbon atoms and at least one carbon tocarbon triple bond. Up to three carbon-carbon triple bonds may bepresent. Thus, “C₂-C₆ alkynyl” means an alkynyl radical having from 2 to6 carbon atoms. Alkynyl groups include ethynyl, propynyl, butynyl,3-methylbutynyl and so on. The straight, branched or cyclic portion ofthe alkynyl group may contain triple bonds and may be substituted if asubstituted alkynyl group is indicated.

In certain instances, substituents may be defined with a range ofcarbons that includes zero, such as (C₀-C₆)alkylene-aryl. If aryl istaken to be phenyl, this definition would include phenyl itself as wellas —CH₂Ph, —CH₂CH₂Ph, CH(CH₃)CH₂CH(CH₃)Ph, and so on.

In one embodiment, as used herein, “aryl” is intended to mean any stablemonocyclic or bicyclic carbon ring of up to 7 atoms in each ring,wherein at least one ring is aromatic. Examples of such aryl elementsinclude phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl. Incases where the aryl substituent is bicyclic and one ring isnon-aromatic, it is understood that attachment is via the aromatic ring.

In another embodiment, “aryl” is an aromatic ring of 5 to 14 carbonsatoms, and includes a carbocyclic aromatic group fused with a 5- or6-membered cycloalkyl group such as indan. Examples of carbocyclicaromatic groups include, but are not limited to, phenyl, naphthyl, e.g.,1-naphthyl and 2-naphthyl; anthracenyl, e.g., 1-anthracenyl,2-anthracenyl; phenanthrenyl; fluorenonyl, e.g., 9-fluorenonyl, indanyland the like. A carbocyclic aromatic group is optionally substitutedwith a designated number of substituents, described below.

The term heteroaryl, as used herein, represents a stable monocyclic orbicyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and contains from 1 to 4 heteroatoms selected from the groupconsisting of O, N and S. In another embodiment, the term heteroarylrefers to a monocyclic, bicyclic or tricyclic aromatic ring of 5- to14-ring atoms of carbon and from one to four heteroatoms selected fromO, N, or S. Heteroaryl groups within the scope of this definitioninclude but are not limited to: acridinyl, carbazolyl, cinnolinyl,quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl,benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl,isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl,pyrrolyl, tetrahydroquinoline. As with the definition of heterocyclebelow, “heteroaryl” is also understood to include the N-oxide derivativeof any nitrogen-containing heteroaryl. In cases where the heteroarylsubstituent is bicyclic and one ring is non-aromatic or contains noheteroatoms, it is understood that attachment is via the aromatic ringor via the heteroatom containing ring, respectively.

In another embodiment, “heteroaryl” is a monocyclic, bicyclic ortricyclic aromatic ring of 5- to 14-ring atoms of carbon and from one tofour heteroatoms selected from O, N, or S. Examples of heteroarylinclude, but are not limited to pyridyl, e.g., 2-pyridyl (also referredto as α-pyridyl), 3-pyridyl (also referred to as β-pyridyl) and4-pyridyl (also referred to as (γ-pyridyl); thienyl, e.g., 2-thienyl and3-thienyl; furanyl, e.g., 2-furanyl and 3-furanyl; pyrimidyl, e.g.,2-pyrimidyl and 4-pyrimidyl; imidazolyl, e.g., 2-imidazolyl; pyranyl,e.g., 2-pyranyl and 3-pyranyl; pyrazolyl, e.g., 4-pyrazolyl and5-pyrazolyl; thiazolyl, e.g., 2-thiazolyl, 4-thiazolyl and 5-thiazolyl;thiadiazolyl; isothiazolyl; oxazolyl, e.g., 2-oxazoyl, 4-oxazoyl and5-oxazoyl; isoxazoyl; pyrrolyl; pyridazinyl; pyrazinyl and the like.Heterocyclic aromatic (or heteroaryl) as defined above may be optionallysubstituted with a designated number of substituents, as described belowfor aromatic groups.

In an embodiment, “heteroaryl” may also include a “fused polycyclicaromatic”, which is a heteroaryl fused with one or more other heteroarylor nonaromatic heterocyclic ring. Examples include, quinolinyl andisoquinolinyl, e.g., 2-quinolinyl, 3-quinolinyl, 4-quinolinyl,5-quinolinyl, 6-quinolinyl, 7-quinolinyl and 8-quinolinyl,1-isoquinolinyl, 3-quinolinyl, 4-isoquinolinyl, 5-isoquinolinyl,6-isoquinolinyl, 7-isoquinolinyl and 8-isoquinolinyl; benzofuranyl,e.g., 2-benzofuranyl and 3-benzofuranyl; dibenzofuranyl, e.g.,2,3-dihydrobenzofuranyl; dibenzothiophenyl; benzothienyl, e.g.,2-benzothienyl and 3-benzothienyl; indolyl, e.g., 2-indolyl and3-indolyl; benzothiazolyl, e.g., 2-benzothiazolyl; benzooxazolyl, e.g.,2-benzooxazolyl; benzimidazolyl, e.g., 2-benzoimidazolyl; isoindolyl,e.g., 1-isoindolyl and 3-isoindolyl; benzotriazolyl; purinyl;thianaphthenyl, pyrazinyl and the like. Fused polycyclic aromatic ringsystems may optionally be substituted with a designated number ofsubstituents, as described herein.

The term “heterocycle” or “heterocyclyl” as used herein is intended tomean a 3- to 10-membered aromatic or nonaromatic heterocycle containingfrom 1 to 4 heteroatoms selected from the group consisting of O, N andS, and includes bicyclic groups. A nonaromatic heterocycle may be fusedwith an aromatic aryl group such as phenyl or aromatic heterocycle.

“Heterocyclyl” therefore includes the above mentioned heteroaryls, aswell as dihydro and tetrahydro analogs thereof. Further examples of“heterocyclyl” include, but are not limited to the following:azetidinyl, benzoimidazolyl, benzofuranyl, benzofurazanyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl,indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl,pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl,tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl,triazolyl, azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl,piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, andN-oxides thereof. Attachment of a heterocyclyl substituent can occur viaa carbon atom or via a heteroatom.

In an embodiment, “heterocycle” (also referred to herein as“heterocyclyl”), is a monocyclic, bicyclic or tricyclic saturated orunsaturated ring of 5- to 14-ring atoms of carbon and from one to fourheteroatoms selected from O, N, S or P. Examples of heterocyclic ringsinclude, but are not limited to: pyrrolidinyl, piperidinyl, morpholinyl,thiamorpholinyl, piperazinyl, dihydrofuranyl, tetrahydrofuranyl,dihydropyranyl, tetrahydrodropyranyl, dihydroquinolinyl,tetrahydroquinolinyl, dihydroisoquinolinyl, tetrahydroisoquinolinyl,dihydropyrazinyl, tetrahydropyrazinyl, dihydropyridyl, tetrahydropyridyland the like.

An “alkylaryl group” (arylalkyl) is an alkyl group substituted with anaromatic group, preferably a phenyl group. A preferred alkylaryl groupis a benzyl group. Suitable aromatic groups are described herein andsuitable alkyl groups are described herein. Suitable substituents for analkylaryl group are described herein.

An “alkylheterocyclyl” group” is an alkyl group substituted with aheterocyclyl group. Suitable heterocyclyl groups are described hereinand suitable alkyl groups are described herein. Suitable substituentsfor an alkyheterocyclyl group are described herein.

An “alkylcycloalkyl group” is an alkyl group substituted with acycloalkyl group. Suitable cycloalkyl groups are described herein andsuitable alkyl groups are described herein. Suitable substituents for analkylcycloalkyl group are described herein.

An “aryloxy group” is an aryl group that is attached to a compound viaan oxygen (e.g., phenoxy).

An “alkoxy group” (alkyloxy), as used herein, is a straight chain orbranched C₁-C₁₂ or cyclic C₃-C₁₂ alkyl group that is connected to acompound via an oxygen atom. Examples of alkoxy groups include but arenot limited to methoxy, ethoxy and propoxy.

An “arylalkoxy group” (arylalkyloxy) is an arylalkyl group that isattached to a compound via an oxygen on the alkyl portion of thearylalkyl (e.g., phenylmethoxy).

An “arylamino group” as used herein, is an aryl group that is attachedto a compound via a nitrogen.

As used herein, an “arylalkylamino group” is an arylalkyl group that isattached to a compound via a nitrogen on the alkyl portion of thearylalkyl.

An “alkylsulfonyl group” as used herein, is an alkyl group that isattached to a compound via the sulfur of a sulfonyl group.

As used herein, many moieties or groups are referred to as being either“substituted or unsubstituted”. When a moiety is referred to assubstituted, it denotes that any portion of the moiety that is known toone skilled in the art as being available for substitution can besubstituted. The phrase “optionally substituted with one or moresubstituents” means one substituent, two substituents, threesubstituents, four substituents or five substituents. For example, thesubstitutable group can be a hydrogen atom that is replaced with a groupother than hydrogen (i.e., a substituent group). Multiple substituentgroups can be present. When multiple substituents are present, thesubstituents can be the same or different and substitution can be at anyof the substitutable sites. Such means for substitution are well knownin the art. For purposes of exemplification, which should not beconstrued as limiting the scope of this invention, some examples ofgroups that are substituents are: alkyl groups (which can also besubstituted, with one or more substituents), alkoxy groups (which can besubstituted), a halogen or halo group (F, Cl, Br, I), hydroxy, nitro,oxo, —CN, —COH, —COOH, amino, azido, N-alkylamino or N,N-dialkylamino(in which the alkyl groups can also be substituted), N-arylamino orN,N-diarylamino (in which the aryl groups can also be substituted),esters (—C(O)—OR, where R can be a group such as alkyl, aryl, etc.,which can be substituted), ureas (—NHC(O)—NHR, where R can be a groupsuch as alkyl, aryl, etc., which can be substituted), carbamates(—NHC(O)—OR, where R can be a group such as alkyl, aryl, etc., which canbe substituted), sulfonamides (—NHS(O)₂R, where R can be a group such asalkyl, aryl, etc., which can be substituted), alkylsulfonyl (which canbe substituted), aryl (which can be substituted), cycloalkyl (which canbe substituted) alkylaryl (which can be substituted), alkylheterocyclyl(which can be substituted), alkylcycloalkyl (which can be substituted),and aryloxy.

It is understood that one or more silicon (Si) atoms can be incorporatedinto the compounds of the instant invention in place of one or morecarbon atoms by one of ordinary skill in the art to provide compoundsthat are chemically stable and that can be readily synthesized bytechniques known in the art from readily available starting materials.Carbon and silicon differ in their covalent radius leading todifferences in bond distance and the steric arrangement when comparinganalogous C-element and Si-element bonds. These differences lead tosubtle changes in the size and shape of silicon-containing compoundswhen compared to carbon. One of ordinary skill in the art wouldunderstand that size and shape differences can lead to subtle ordramatic changes in potency, solubility, lack of off target activity,packaging properties, and so on. (Diass, J. O. et al. Organometallics(2006) 5:1188-1198; Showell, G. A. et al. Bioorganic & MedicinalChemistry Letters (2006) 16:2555-2558).

Stereochemistry

Many organic compounds exist in optically active forms having theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L or R and S are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and l or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesecompounds, called stereoisomers, are identical except that they arenon-superimposable mirror images of one another. A specific stereoisomercan also be referred to as an enantiomer, and a mixture of such isomersis often called an enantiomeric mixture. A 50:50 mixture of enantiomersis referred to as a racemic mixture. Many of the compounds describedherein can have one or more chiral centers and therefore can exist indifferent enantiomeric forms. If desired, a chiral carbon can bedesignated with an asterisk (*). When bonds to the chiral carbon aredepicted as straight lines in the Formulas of the invention, it isunderstood that both the (R) and (S) configurations of the chiralcarbon, and hence both enantiomers and mixtures thereof, are embracedwithin the Formula. As is used in the art, when it is desired to specifythe absolute configuration about a chiral carbon, one of the bonds tothe chiral carbon can be depicted as a wedge (bonds to atoms above theplane) and the other can be depicted as a series or wedge of shortparallel lines is (bonds to atoms below the plane). TheCahn-Inglod-Prelog system can be used to assign the (R) or (S)configuration to a chiral carbon.

When the HDAC inhibitors of the present invention contain one chiralcenter, the compounds exist in two enantiomeric forms and the presentinvention includes both enantiomers and mixtures of enantiomers, such asthe specific 50:50 mixture referred to as a racemic mixtures. Theenantiomers can be resolved by methods known to those skilled in theart, such as formation of diastereoisomeric salts which may beseparated, for example, by crystallization (see, CRC Handbook of OpticalResolutions via Diastereomeric Salt Formation by David Kozma (CRC Press,2001)); formation of diastereoisomeric derivatives or complexes whichmay be separated, for example, by crystallization, gas-liquid or liquidchromatography; selective reaction of one enantiomer with anenantiomer-specific reagent, for example enzymatic esterification; orgas-liquid or liquid chromatography in a chiral environment, for exampleon a chiral support for example silica with a bound chiral ligand or inthe presence of a chiral solvent. It will be appreciated that where thedesired enantiomer is converted into another chemical entity by one ofthe separation procedures described above, a further step is required toliberate the desired enantiomeric form. Alternatively, specificenantiomers may be synthesized by asymmetric synthesis using opticallyactive reagents, substrates, catalysts or solvents, or by converting oneenantiomer into the other by asymmetric transformation.

Designation of a specific absolute configuration at a chiral carbon ofthe compounds of the invention is understood to mean that the designatedenantiomeric form of the compounds is in enantiomeric excess (ee) or inother words is substantially free from the other enantiomer. Forexample, the “R” forms of the compounds are substantially free from the“S” forms of the compounds and are, thus, in enantiomeric excess of the“S” forms. Conversely, “S” forms of the compounds are substantially freeof “R” forms of the compounds and are, thus, in enantiomeric excess ofthe “R” forms. Enantiomeric excess, as used herein, is the presence of aparticular enantiomer at greater than 50%. In a particular embodimentwhen a specific absolute configuration is designated, the enantiomericexcess of depicted compounds is at least about 90%.

When a compound of the present invention has two or more chiral carbonsit can have more than two optical isomers and can exist indiastereoisomeric forms. For example, when there are two chiral carbons,the compound can have up to 4 optical isomers and 2 pairs of enantiomers((S,S)/(R,R) and (R,S)/(S,R)). The pairs of enantiomers (e.g.,(S,S)/(R,R)) are mirror image stereoisomers of one another. Thestereoisomers that are not mirror-images (e.g., (S,S) and (R,S)) arediastereomers. The diastereoisomeric pairs may be separated by methodsknown to those skilled in the art, for example chromatography orcrystallization and the individual enantiomers within each pair may beseparated as described above. The present invention includes eachdiastereoisomer of such compounds and mixtures thereof.

As used herein, “a,” an” and “the” include singular and plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “an active agent” or “a pharmacologically active agent”includes a single active agent as well a two or more different activeagents in combination, reference to “a carrier” includes mixtures of twoor more carriers as well as a single carrier, and the like.

This invention is also intended to encompass pro-drugs of the compoundsof the instant invention disclosed herein. A prodrug of any of thecompounds can be made using well-known pharmacological techniques.

This invention, in addition to the above listed compounds, is intendedto encompass the use of homologs and analogs of such compounds. In thiscontext, homologs are molecules having substantial structuralsimilarities to the above-described compounds and analogs are moleculeshaving substantial biological similarities regardless of structuralsimilarities.

Pharmaceutically Acceptable Salts

The compounds of the instant invention described herein can, as notedabove, be prepared in the form of their pharmaceutically acceptablesalts. Pharmaceutically acceptable salts are salts that retain thedesired biological activity of the parent compound and do not impartundesired toxicological effects. Examples of such salts are acidaddition salts, organic and inorganic acids, for example, acid additionsalts which may, for example, be hydrochloric acid, sulphuric acid,methanesulphonic acid, fumaric acid, maleic acid, succinic acid, aceticacid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonicacid, trifluoroacetic acid, formic acid, phosphoric acid and the like.Pharmaceutically acceptable salts can also be prepared from by treatmentwith inorganic bases, for example, sodium, potassium, ammonium, calcium,or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.Pharmaceutically acceptable salts can also salts formed from elementalanions such as chlorine, bromine and iodine.

The active compounds disclosed can, as noted above, also be prepared inthe form of their hydrates. The term “hydrate” includes but is notlimited to hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrateand the like.

The active compounds disclosed can, as noted above, also be prepared inthe form of a solvate with any organic or inorganic solvent, for examplealcohols such as methanol, ethanol, propanol and isopropanol, ketonessuch as acetone, aromatic solvents and the like.

The active compounds disclosed can also be prepared in any solid orliquid physical form. For example, the compound can be in a crystallineform, in amorphous form, and have any particle size. Furthermore, thecompound particles may be micronized, or may be agglomerated,particulate granules, powders, oils, oily suspensions or any other formof solid or liquid physical form.

The compounds of the present invention may also exhibit polymorphism.This invention further includes different polymorphs of the compounds ofthe present invention. The term “polymorph” refers to a particularcrystalline state of a substance, having particular physical propertiessuch as X-ray diffraction, IR spectra, melting point, and the like.

As used herein, “a,” an” and “the” include singular and plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “an active agent” or “a pharmacologically active agent”includes a single active agent as well a two or more different activeagents in combination, reference to “a carrier” includes mixtures of twoor more carriers as well as a single carrier, and the like.

Methods of Treatment

The invention also relates to methods of using the compounds of theinstant invention. As demonstrated herein, the compounds of the presentinvention are useful for the treatment of cancer. In addition, there isa wide range of other diseases for which substituted nicotinamides maybe useful. Non-limiting examples are thioredoxin (TRX)-mediated diseasesas described herein, and diseases of the central nervous system (CNS) asdescribed herein.

1. Treatment of Cancer

As demonstrated herein, the compounds of the present invention areuseful for the treatment of cancer. Accordingly, in one embodiment, theinvention relates to a method of treating cancer in a subject in need oftreatment comprising administering to said subject a therapeuticallyeffective amount of the compounds of the instant invention.

The term “cancer” refers to any cancer caused by the proliferation ofneoplastic cells, such as solid tumors, neoplasms, carcinomas, sarcomas,leukemias, lymphomas and the like. In particular, cancers that may betreated by the compounds, compositions and methods of the inventioninclude, but are not limited to: Cardiac: sarcoma (angiosarcoma,fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma,fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamouscell, undifferentiated small cell, undifferentiated large cell,adenocarcinoma, non-small cell lung), alveolar (bronchiolar) carcinoma,bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma,mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma,adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma), colon, rectal, colorectal; Genitourinary tract: kidney(adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia),bladder and urethra (squamous cell carcinoma, transitional cellcarcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis(seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.Thus, the term “cancerous cell” as provided herein, includes a cellafflicted by any one of the above-identified conditions.

In an embodiment, the instant compounds are useful in the treatment ofcancers that include, but are not limited to: leukemias including acuteleukemias and chronic leukemias such as acute lymphocytic leukemia(ALL), Acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL),chronic myelogenous leukemia (CML) and Hairy Cell Leukemia; lymphomassuch as cutaneous T-cell lymphomas (CTCL), noncutaneous peripheralT-cell lymphomas, lymphomas associated with human T-cell lymphotrophicvirus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), Hodgkin'sdisease and non-Hodgkin's lymphomas, large-cell lymphomas, diffuse largeB-cell lymphoma (DLBCL); Burkitt's lymphoma; mesothelioma, primarycentral nervous system (CNS) lymphoma; multiple myeloma; childhood solidtumors such as brain tumors, neuroblastoma, retinoblastoma, Wilm'stumor, bone tumors, and soft-tissue sarcomas, common solid tumors ofadults such as head and neck cancers (e.g., oral, laryngeal andesophageal), genito urinary cancers (e.g., prostate, bladder, renal,uterine, ovarian, testicular, rectal and colon), lung cancer, breastcancer, pancreatic cancer, melanoma and other skin cancers, stomachcancer, brain tumors, liver cancer and thyroid cancer.

2. Treatment of Thioredoxin (TRX)-Mediated Diseases

In another embodiment, the compounds of the instant invention are usedin a method of treating a thioredoxin (TRX)-mediated disease or disorderin a subject in need thereof, comprising administering to the subject atherapeutically effective amount of one or more of the compounds of theinstant invention.

Examples of TRX-mediated diseases include, but are not limited to, acuteand chronic inflammatory diseases, autoimmune diseases, allergicdiseases, diseases associated with oxidative stress, and diseasescharacterized by cellular hyperproliferation.

Non-limiting examples are inflammatory conditions of a joint includingrheumatoid arthritis (RA) and psoriatic arthritis; inflammatory boweldiseases such as Crohn's disease and ulcerative colitis;spondyloarthropathies; scleroderma; psoriasis (including T-cell mediatedpsoriasis) and inflammatory dermatoses such an dermatitis, eczema,atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis(e.g., necrotizing, cutaneous, and hypersensitivity vasculitis);eosinphilic myositis, eosinophilic fasciitis; cancers with leukocyteinfiltration of the skin or organs, ischemic injury, including cerebralischemia (e.g., brain injury as a result of trauma, epilepsy, hemorrhageor stroke, each of which may lead to neurodegeneration); HIV, heartfailure, chronic, acute or malignant liver disease, autoimmunethyroiditis; systemic lupus erythematosus, Sjorgren's syndrome, lungdiseases (e.g., ARDS); acute pancreatitis; amyotrophic lateral sclerosis(ALS); Alzheimer's disease; cachexia/anorexia; asthma; atherosclerosis;chronic fatigue syndrome, fever; diabetes (e.g., insulin diabetes orjuvenile onset diabetes); glomerulonephritis; graft versus hostrejection (e.g., in transplantation); hemohorragic shock; hyperalgesia:inflammatory bowel disease; multiple sclerosis; myopathies (e.g., muscleprotein metabolism, esp. in sepsis); osteoporosis; Parkinson's disease;pain; pre-term labor; psoriasis; reperfusion injury; cytokine-inducedtoxicity (e.g., septic shock, endotoxic shock); side effects fromradiation therapy, temporal mandibular joint disease, tumor metastasis;or an inflammatory condition resulting from strain, sprain, cartilagedamage, trauma such as burn, orthopedic surgery, infection or otherdisease processes. Allergic diseases and conditions, include but are notlimited to respiratory allergic diseases such as asthma, allergicrhinitis, hypersensitivity lung diseases, hypersensitivity pneumonitis,eosinophilic pneumonias (e.g., Loeffler's syndrome, chronic eosinophilicpneumonia), delayed-type hypersensitivity, interstitial lung diseases(ILD) (e.g., idiopathic pulmonary fibrosis, or ILD associated withrheumatoid arthritis, systemic lupus erythematosus, ankylosingspondylitis, systemic sclerosis, Sjogren's syndrome, polymyositis ordermatomyositis); systemic anaphylaxis or hypersensitivity responses,drug allergies (e.g., to penicillin, cephalosporins), insect stingallergies, and the like.

3. Treatment of Diseases of the Central Nervous System (CNS)

In another embodiment, the compounds of the instant invention are usedin a method of treating a disease of the central nervous system in asubject in need thereof comprising administering to the subject atherapeutically effective amount of any one or more of the compounds ofthe instant invention.

In a particular embodiment, the CNS disease is a neurodegenerativedisease. In a further embodiment, the neurodegenerative disease is aninherited neurodegenerative disease, such as those inheritedneurodegenerative diseases that are polyglutamine expansion diseases.Generally, neurodegenerative diseases can be grouped as follows:

I. Disorders characterized by progressive dementia in the absence ofother prominent neurologic signs, such as Alzheimer's disease; Seniledementia of the Alzheimer type; and Pick's disease (lobar atrophy).

II. Syndromes combining progressive dementia with other prominentneurologic abnormalities such as A) syndromes appearing mainly in adults(e.g., Huntington's disease, Multiple system atrophy combining dementiawith ataxia and/or manifestations of Parkinson's disease, Progressivesupranuclear palsy (Steel-Richardson-Olszewski), diffuse Lewy bodydisease, and corticodentatonigral degeneration); and B) syndromesappearing mainly in children or young adults (e.g., Hallervorden-Spatzdisease and progressive familial myoclonic epilepsy).

III. Syndromes of gradually developing abnormalities of posture andmovement such as paralysis agitans (Parkinson's disease), striatonigraldegeneration, progressive supranuclear palsy, torsion dystonia (torsionspasm; dystonia musculorum deformans), spasmodic torticollis and otherdyskinesis, familial tremor, and Gilles de la Tourette syndrome.

IV. Syndromes of progressive ataxia such as cerebellar degenerations(e.g., cerebellar cortical degeneration and olivopontocerebellar atrophy(OPCA)); and spinocerebellar degeneration (Friedreich's atazia andrelated disorders).

V. Syndrome of central autonomic nervous system failure (Shy-Dragersyndrome).

VI. Syndromes of muscular weakness and wasting without sensory changes(motorneuron disease such as amyotrophic lateral sclerosis, spinalmuscular atrophy (e.g., infantile spinal muscular atrophy(Werdnig-Hoffman), juvenile spinal muscular atrophy(Wohlfart-Kugelberg-Welander) and other forms of familial spinalmuscular atrophy), primary lateral sclerosis, and hereditary spasticparaplegia.

VII. Syndromes combining muscular weakness and wasting with sensorychanges (progressive neural muscular atrophy; chronic familialpolyneuropathies) such as peroneal muscular atrophy(Charcot-Marie-Tooth), hypertrophic interstitial polyneuropathy(Dejerine-Sottas), and miscellaneous forms of chronic progressiveneuropathy.

VIII. Syndromes of progressive visual loss such as pigmentarydegeneration of the retina (retinitis pigmentosa), and hereditary opticatrophy (Leber's disease).

DEFINITIONS

The term “treating” in its various grammatical forms in relation to thepresent invention refers to preventing (i.e., chemoprevention), curing,reversing, attenuating, alleviating, minimizing, suppressing or haltingthe deleterious effects of a disease state, disease progression, diseasecausative agent (e.g., bacteria or viruses) or other abnormal condition.For example, treatment may involve alleviating a symptom (i.e., notnecessary all symptoms) of a disease or attenuating the progression of adisease. Because some of the inventive methods involve the physicalremoval of the etiological agent, the artisan will recognize that theyare equally effective in situations where the inventive compound isadministered prior to, or simultaneous with, exposure to the etiologicalagent (prophylactic treatment) and situations where the inventivecompounds are administered after (even well after) exposure to theetiological agent.

Treatment of cancer, as used herein, refers to partially or totallyinhibiting, delaying or preventing the progression of cancer includingcancer metastasis; inhibiting, delaying or preventing the recurrence ofcancer including cancer metastasis; or preventing the onset ordevelopment of cancer (chemoprevention) in a mammal, for example ahuman.

As used herein, the term “therapeutically effective amount” is intendedto encompass any amount that will achieve the desired therapeutic orbiological effect. The therapeutic effect is dependent upon the diseaseor disorder being treated or the biological effect desired. As such, thetherapeutic effect can be a decrease in the severity of symptomsassociated with the disease or disorder and/or inhibition (partial orcomplete) of progression of the disease. The amount needed to elicit thetherapeutic response can be determined based on the age, health, sizeand sex of the subject. Optimal amounts can also be determined based onmonitoring of the subject's response to treatment.

In the present invention, when the compounds are used to treat orprevent cancer, the desired biological response is partial or totalinhibition, delay or prevention of the progression of cancer includingcancer metastasis; inhibition, delay or prevention of the recurrence ofcancer including cancer metastasis; or the prevention of the onset ordevelopment of cancer (chemoprevention) in a mammal, for example ahuman.

Furthermore, in the present invention, when the compounds are used totreat and/or prevent thioredoxin (TRX)-mediated diseases and conditions,a therapeutically effective amount is an amount that regulates, forexample, increases, decreases or maintains a physiologically suitablelevel of TRX in the subject in need of treatment to elicit the desiredtherapeutic effect. The therapeutic effect is dependent upon thespecific TRX-mediated disease or condition being treated. As such, thetherapeutic effect can be a decrease in the severity of symptomsassociated with the disease or disorder and/or inhibition (partial orcomplete) of progression of the disease or disease.

Furthermore, in the present invention, when the compounds are used totreat and/or prevent diseases or disorders of the central nervous system(CNS), a therapeutically effective amount is dependent upon the specificdisease or disorder being treated. As such, the therapeutic effect canbe a decrease in the severity of symptoms associated with the disease ordisorder and/or inhibition (partial or complete) of progression of thedisease or disorder.

In addition, a therapeutically effective amount can be an amount thatinhibits histone deacetylase.

Further, a therapeutically effective amount can be an amount thatselectively induces terminal differentiation, cell growth arrest and/orapoptosis of neoplastic cells, or an amount that induces terminaldifferentiation of tumor cells.

The method of the present invention is intended for the treatment orchemoprevention of human patients with cancer. However, it is alsolikely that the method would be effective in the treatment of cancer inother subjects. “Subject”, as used herein, refers to animals such asmammals, including, but not limited to, primates (e.g., humans), cows,sheep, goats, horses, pigs, dogs, cats, rabbits, guinea pigs, rats, miceor other bovine, ovine, equine, canine, feline, rodent or murinespecies.

Histone Deacetylases and Histone Deacetylase Inhibitors

As demonstrated herein, the compounds of the present invention showimproved activity as histone deacetylase (HDAC) inhibitors. Accordingly,in one embodiment, the invention relates to a method of inhibiting theactivity of histone deacetylase comprising contacting the histonedeacetylase with an effective amount of one or more of the compounds ofthe instant invention.

Histone deacetylases (HDACs), as that term is used herein, are enzymesthat catalyze the removal of acetyl groups from lysine residues in theamino terminal tails of the nucleosomal core histones. As such, HDACstogether with histone acetyl transferases (HATs) regulate theacetylation status of histones. Histone acetylation affects geneexpression and inhibitors of HDACs, such as the hydroxamic acid-basedhybrid polar compound suberoylanilide hydroxamic acid (SAHA) inducegrowth arrest, differentiation and/or apoptosis of transformed cells invitro and inhibit tumor growth in vivo. HDACs can be divided into threeclasses based on structural homology. Class I HDACs (HDACs 1, 2, 3 and8) bear similarity to the yeast RPD3 protein, are located in the nucleusand are found in complexes associated with transcriptionalco-repressors. Class II HDACs (HDACs 4, 5, 6, 7 and 9) are similar tothe yeast HDA1 protein, and have both nuclear and cytoplasmicsubcellular localization. Both Class I and II HDACs are inhibited byhydroxamic acid-based HDAC inhibitors, such as SAHA. Class III HDACsform a structurally distant class of NAD dependent enzymes that arerelated to the yeast SIR2 proteins and are not inhibited by hydroxamicacid-based HDAC inhibitors.

Histone deacetylase inhibitors or HDAC inhibitors, as that term is usedherein are compounds that are capable of inhibiting the deacetylation ofhistones in vivo, in vitro or both. As such, HDAC inhibitors inhibit theactivity of at least one histone deacetylase. As a result of inhibitingthe deacetylation of at least one histone, an increase in acetylatedhistone occurs and accumulation of acetylated histone is a suitablebiological marker for assessing the activity of HDAC inhibitors.Therefore, procedures that can assay for the accumulation of acetylatedhistones can be used to determine the HDAC inhibitory activity ofcompounds of interest. It is understood that compounds that can inhibithistone deacetylase activity can also bind to other substrates and assuch can inhibit other biologically active molecules such as enzymes. Itis also to be understood that the compounds of the present invention arecapable of inhibiting any of the histone deacetylases set forth above,or any other histone deacetylases.

For example, in patients receiving HDAC inhibitors, the accumulation ofacetylated histones in peripheral mononuclear cells as well as in tissuetreated with HDAC inhibitors can be determined against a suitablecontrol.

HDAC inhibitory activity of a particular compound can be determined invitro using, for example, an enzymatic assays which shows inhibition ofat least one histone deacetylase. Further, determination of theaccumulation of acetylated histones in cells treated with a particularcomposition can be determinative of the HDAC inhibitory activity of acompound.

Assays for the accumulation of acetylated histones are well known in theliterature. See, for example, Marks, P. A. et al., J. Natl. CancerInst., 92:1210-1215, 2000, Butler, L. M. et al., Cancer Res.60:5165-5170 (2000), Richon, V. M. et al., Proc. Natl. Acad. Sci., USA,95:3003-3007, 1998, and Yoshida, M. et al., J. Biol. Chem.,265:17174-17179, 1990.

For example, an enzymatic assay to determine the activity of an HDACinhibitor compound can be conducted as follows. Briefly, the effect ofan HDAC inhibitor compound on affinity purified human epitope-tagged(Flag) HDAC1 can be assayed by incubating the enzyme preparation in theabsence of substrate on ice for about 20 minutes with the indicatedamount of inhibitor compound. Substrate ([³H]acetyl-labelled murineerythroleukemia cell-derived histone) can be added and the sample can beincubated for 20 minutes at 37° C. in a total volume of 30 μL. Thereaction can then be stopped and released acetate can be extracted andthe amount of radioactivity release determined by scintillationcounting. An alternative assay useful for determining the activity of anHDAC inhibitor compound is the “HDAC Fluorescent Activity Assay; DrugDiscovery Kit-AK-500” available from BIOMOL Research Laboratories, Inc.,Plymouth Meeting, Pa.

In vivo studies can be conducted as follows. Animals, for example, mice,can be injected intraperitoneally with an HDAC inhibitor compound.Selected tissues, for example, brain, spleen, liver etc, can be isolatedat predetermined times, post administration. Histones can be isolatedfrom tissues essentially as described by Yoshida et al., J. Biol. Chem.265:17174-17179, 1990. Equal amounts of histones (about 1 μg) can beelectrophoresed on 15% SDS-polyacrylamide gels and can be transferred toHybond-P filters (available from Amersham). Filters can be blocked with3% milk and can be probed with a rabbit purified polyclonalanti-acetylated histone H4 antibody (αAc-H4) and anti-acetylated histoneH3 antibody (αAc-H3) (Upstate Biotechnology, Inc.). Levels of acetylatedhistone can be visualized using a horseradish peroxidase-conjugated goatanti-rabbit antibody (1:5000) and the SuperSignal chemiluminescentsubstrate (Pierce). As a loading control for the histone protein,parallel gels can be run and stained with Coomassie Blue (CB).

In addition, hydroxamic acid-based HDAC inhibitors have been shown to upregulate the expression of the p21^(WAF1) gene. The p21^(WAF1) proteinis induced within 2 hours of culture with HDAC inhibitors in a varietyof transformed cells using standard methods. The induction of thep21^(WAF1) gene is associated with accumulation of acetylated histonesin the chromatin region of this gene. Induction of p21^(WAF1) cantherefore be recognized as involved in the G1 cell cycle arrest causedby HDAC inhibitors in transformed cells.

Combination Therapy

The compounds of the present invention can be administered alone or incombination with other therapies suitable for the disease or disorderbeing treated. Where separate dosage formulations are used, thecompounds of the instant invention and the other therapeutic agent canbe administered at essentially the same time (concurrently) or atseparately staggered times (sequentially). The pharmaceuticalcombination is understood to include all these regimens. Administrationin these various ways are suitable for the present invention as long asthe beneficial therapeutic effect of the compounds of the instantinvention and the other therapeutic agent are realized by the patient atsubstantially the same time. In an embodiment, such beneficial effect isachieved when the target blood level concentrations of each active drugare maintained at substantially the same time.

The instant compounds may also be useful in combination with knowntherapeutic agents and anti-cancer agents. For example, instantcompounds are useful in combination with known anti-cancer agents.Combinations of the presently disclosed compounds with other anti-canceror chemotherapeutic agents are within the scope of the invention.Examples of such agents can be found in Cancer Principles and Practiceof Oncology by V. T. Devita and S. Hellman (editors), 6^(th) edition(Feb. 15, 2001), Lippincott Williams & Wilkins Publishers. A person ofordinary skill in the art would be able to discern which combinations ofagents would be useful based on the particular characteristics of thedrugs and the cancer involved. Such anti-cancer agents include, but arenot limited to, the following: estrogen receptor modulators, androgenreceptor modulators, retinoid receptor modulators, cytotoxic/cytostaticagents, antiproliferative agents, prenyl-protein transferase inhibitors,HMG-CoA reductase inhibitors and other angiogenesis inhibitors,inhibitors of cell proliferation and survival signaling, apoptosisinducing agents, agents that interfere with cell cycle checkpoints,agents that interfere with receptor tyrosine kinases (RTKs) and cancervaccines. The instant compounds are particularly useful whenco-administered with radiation therapy.

In an embodiment, the instant compounds may also be useful incombination with known anti-cancer agents including the following:estrogen receptor modulators, androgen receptor modulators, retinoidreceptor modulators, cytotoxic agents, antiproliferative agents,prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIVprotease inhibitors, reverse transcriptase inhibitors, and otherangiogenesis inhibitors.

“Estrogen receptor modulators” refers to compounds that interfere withor inhibit the binding of estrogen to the receptor, regardless ofmechanism. Examples of estrogen receptor modulators include, but are notlimited to, diethylstibestral, tamoxifen, raloxifene, idoxifene,LY353381, LY117081, toremifene, fluoxymestero, lfulvestrant,4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

Other hormonal agents include: aromatase inhibitors (e.g.,aminoglutethimide, anastrozole and tetrazole), luteinizing hormonerelease hormone (LHRH) analogues, ketoconazole, goserelin acetate,leuprolide, megestrol acetate and mifepristone.

“Androgen receptor modulators” refers to compounds which interfere orinhibit the binding of androgens to the receptor, regardless ofmechanism. Examples of androgen receptor modulators include finasterideand other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide,liarozole, and abiraterone acetate.

“Retinoid receptor modulators” refers to compounds which interfere orinhibit the binding of retinoids to the receptor, regardless ofmechanism. Examples of such retinoid receptor modulators includebexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid,α-difluoromethyl-ornithine, ILX23-7553,trans-N-(4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.

“Cytotoxic/cytostatic agents” refer to compounds which cause cell deathor inhibit cell proliferation primarily by interfering directly with thecell's functioning or inhibit or interfere with cell mytosis, includingalkylating agents, tumor necrosis factors, intercalators, hypoxiaactivatable compounds, microtubule inhibitors/microtubule-stabilizingagents, inhibitors of mitotic kinesins, inhibitors of histonedeacetylase, inhibitors of kinases involved in mitotic progression,antimetabolites; biological response modifiers; hormonal/anti-hormonaltherapeutic agents, haematopoietic growth factors, monoclonal antibodytargeted therapeutic agents, topoisomerase inhibitors, proteasomeinhibitors and ubiquitin ligase inhibitors.

Examples of cytotoxic agents include, but are not limited to, sertenef,cachectin, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine,melphalan, uracil mustard, thiotepa, busulfan, carmustine, lomustine,streptozocin, tasonermin, lonidamine, carboplatin, altretamine,dacarbazine, procarbazine, prednimustine, dibromodulcitol, ranimustine,fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin,estramustine, improsulfan tosilate, trofosfamide, nimustine,dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin,cisplatin, irofulven, dexifosfamide,cis-aminedichloro(2-methyl-pyridine)platinum, benzylguanine,glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum(II)]tetrachloride, diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,doxorubicin, daunorubicin, idarubicin, anthracenedione, bleomycin,mitomycin C, dactinomycin, plicatomycin, bisantrene, mitoxantrone,pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycanninomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (seeWO 00/50032).

An example of a hypoxia activatable compound is tirapazamine.

Examples of proteasome inhibitors include but are not limited tolactacystin and bortezomib.

Examples of microtubule inhibitors/microtubule-stabilising agentsinclude vincristine, vinblastine, vindesine, vinzolidine, vinorelbine,vindesine sulfate, 3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine,podophyllotoxins (e.g., etoposide (VP-16) and teniposide (VM-26)),paclitaxel, docetaxol, rhizoxin, dolastatin, mivobulin isethionate,auristatin, cemadotin, RPR109881, BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and6,288,237) and BMS188797.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine,irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione,lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350,BNPI100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide,asulacrine,(5a,5aB,8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroxy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium,6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide,N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one,and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the humanmitotic kinesin KSP, are described in PCT Publications WO 01/30768, WO01/98278, WO 03/050,064, WO 03/050,122, WO 03/049,527, WO 03/049,679, WO03/049,678 and WO 03/39460 and pending PCT Appl. Nos. US03/06403 (filedMar. 4, 2003), US03/15861 (filed May 19, 2003), US03/15810 (filed May19, 2003), US03/18482 (filed Jun. 12, 2003) and US03/18694 (filed Jun.12, 2003). In an embodiment inhibitors of mitotic kinesins include, butare not limited to inhibitors of KSP, inhibitors of MKLP1, inhibitors ofCENP-E, inhibitors of MCAK, inhibitors of Kif14, inhibitors of Mphosph1and inhibitors of Rab6-KIFL.

Examples of “histone deacetylase inhibitors” include, but are notlimited to, SAHA, TSA, oxamflatin, PXD101, MG98, valproic acid andscriptaid. Further reference to other histone deacetylase inhibitors maybe found in the following manuscript; Miller, T. A. et al. J. Med. Chem.46 (24):5097-5116 (2003).

“Inhibitors of kinases involved in mitotic progression” include, but arenot limited to, inhibitors of aurora kinase, inhibitors of Polo-likekinases (PLK; in particular inhibitors of PLK-1), inhibitors of bub-1and inhibitors of bub-R1. An example of an “aurora kinase inhibitor” isVX-680.

“Antiproliferative agents” includes antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-fluorouracil, floxuridine, methotrexate,leucovarin, hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP),cytarabine, pentostatin, fludarabine phosphate, cladribine (2-CDA),asparaginase, gemcitabine, alanosine,1′-acetyl-8-(carbamoyloxy-methyl)-4-formyl-6-methoxy-14-oxa-1,1′-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine and3-aminopyridine-2-carboxaldehyde thiosemicarbazone.

Examples of monoclonal antibody targeted therapeutic agents includethose therapeutic agents which have cytotoxic agents or radioisotopesattached to a cancer cell specific or target cell specific monoclonalantibody. Examples include Bexxar.

“HMG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. Examples of HMG-CoA reductaseinhibitors that may be used include but are not limited to lovastatin(MEVACOR®; see U.S. Pat. Nos. 4,231,938, 4,294,926 and 4,319,039),simvastatin (ZOCOR®; see U.S. Pat. Nos. 4,444,784, 4,820,850 and4,916,239), pravastatin (PRAVACHOL®; see U.S. Pat. Nos. 4,346,227,4,537,859, 4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL®;see U.S. Pat. Nos. 5,354,772, 4,911,165, 4,929,437, 5,189,164,5,118,853, 5,290,946 and 5,356,896) and atorvastatin (LIPITOR®; see U.S.Pat. Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952). The structuralformulas of these and additional HMG-CoA reductase inhibitors that maybe used in the instant methods are described at page 87 of M. Yalpani,“Cholesterol Lowering Drugs”, Chemistry & Industry, pp. 85-89 (5 Feb.1996) and U.S. Pat. Nos. 4,782,084 and 4,885,314. The term HMG-CoAreductase inhibitor as used herein includes all pharmaceuticallyacceptable lactone and open-acid forms (i.e., where the lactone ring isopened to form the free acid) as well as salt and ester forms ofcompounds which have HMG-CoA reductase inhibitory activity, and thereforthe use of such salts, esters, open-acid and lactone forms is includedwithin the scope of this invention.

“Prenyl-protein transferase inhibitor” refers to a compound whichinhibits any one or any combination of the prenyl-protein transferaseenzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-I), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also called RabGGPTase).

Examples of prenyl-protein transferase inhibitors can be found in thefollowing publications and patents: WO 96/30343, WO 97/18813, WO97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO95/32987, U.S. Pat. No. 5,420,245, U.S. Pat. No. 5,523,430, U.S. Pat.No. 5,532,359, U.S. Pat. No. 5,510,510, U.S. Pat. No. 5,589,485, U.S.Pat. No. 5,602,098, European Patent Publ. 0 618 221, European PatentPubl. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ.0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO 95/10515, WO95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO 96/33159, WO96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO98/02436, and U.S. Pat. No. 5,532,359. For an example of the role of aprenyl-protein transferase inhibitor on angiogenesis see European J. ofCancer, Vol. 35, No. 9, pp. 1394-1401 (1999).

“Angiogenesis inhibitors” refers to compounds that inhibit the formationof new blood vessels, regardless of mechanism. Examples of angiogenesisinhibitors include, but are not limited to, tyrosine kinase inhibitors,such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) andFlk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived,or platelet derived growth factors, MMP (matrix metalloprotease)inhibitors, integrin blockers, interferon-α, interleukin-12,erythropoietin (epoietin-α), granulocyte-CSF (filgrastin), granulocyte,macrophage-CSF (sargramostim), pentosan polysulfate, cyclooxygenaseinhibitors, including nonsteroidal anti-inflammatories (NSAIDs) likeaspirin and ibuprofen as well as selective cyclooxy-genase-2 inhibitorslike celecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol.69, p. 475 (1982); Arch. Opthalmol., Vol. 108, p. 573 (1990); Anat.Rec., Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995);Clin, Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p.107 (1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res.,Vol. 57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol.Med., Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)),steroidal anti-inflammatories (such as corticosteroids,mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred,betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab.Clin. Med. 105:141-145 (1985)), and antibodies to VEGF (see, NatureBiotechnology, Vol. 17, pp. 963-968 (October 1999); Kim et al., Nature,362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other therapeutic agents that modulate or inhibit angiogenesis and mayalso be used in combination with the compounds of the instant inventioninclude agents that modulate or inhibit the coagulation and fibrinolysissystems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examplesof such agents that modulate or inhibit the coagulation and fibrinolysispathways include, but are not limited to, heparin (see Thromb. Haemost.80:10-23 (1998)), low molecular weight heparins and carboxypeptidase Uinhibitors (also known as inhibitors of active thrombin activatablefibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354(2001)). TAFIa inhibitors have been described in PCT Publication WO03/013,526 and U.S. Ser. No. 60/349,925 (filed Jan. 18, 2002).

“Agents that interfere with cell cycle checkpoints” refer to compoundsthat inhibit protein kinases that transduce cell cycle checkpointsignals, thereby sensitizing the cancer cell to DNA damaging agents.Such agents include inhibitors of ATR, ATM, the Chk1 and Chk2 kinasesand cdk and cdc kinase inhibitors and are specifically exemplified by7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

“Agents that interfere with receptor tyrosine kinases (RTKs)” refer tocompounds that inhibit RTKs and therefore mechanisms involved inoncogenesis and tumor progression. Such agents include inhibitors ofc-Kit, Eph, PDGF, Flt3 and c-Met. Further agents include inhibitors ofRTKs shown as described by Burne-Jensen and Hunter, Nature, 411:355-365,2001.

“Inhibitors of cell proliferation and survival signaling pathway” referto pharmaceutical agents that inhibit cell surface receptors and signaltransduction cascades downstream of those surface receptors. Such agentsinclude inhibitors of inhibitors of EGFR (for example gefitinib anderlotinib), inhibitors of ERB-2 (for example trastuzumab), inhibitors ofIGFR, inhibitors of CD20 (rituximab), inhibitors of cytokine receptors,inhibitors of MET, inhibitors of PI3K (for example LY294002),serine/threonine kinases (including but not limited to inhibitors of Aktsuch as described in (WO 03/086404, WO 03/086403, WO 03/086394, WO03/086279, WO 02/083675, WO 02/083139, WO 02/083140 and WO 02/083138),inhibitors of Raf kinase (for example BAY-43-9006), inhibitors of MEK(for example CI-1040 and PD-098059) and inhibitors of mTOR (for exampleWyeth CCI-779 and Ariad AP23573). Such agents include small moleculeinhibitor compounds and antibody antagonists.

“Apoptosis inducing agents” include activators of TNF receptor familymembers (including the TRAIL receptors).

The invention also encompasses combinations with NSAID's which areselective COX-2 inhibitors. For purposes of this specification NSAID'swhich are selective inhibitors of COX-2 are defined as those whichpossess a specificity for inhibiting COX-2 over COX-1 of at least 100fold as measured by the ratio of IC₅₀ for COX-2 over IC₅₀ for COX-1evaluated by cell or microsomal assays. Such compounds include, but arenot limited to those disclosed in U.S. Pat. No. 5,474,995, U.S. Pat. No.5,861,419, U.S. Pat. No. 6,001,843, U.S. Pat. No. 6,020,343, U.S. Pat.No. 5,409,944, U.S. Pat. No. 5,436,265, U.S. Pat. No. 5,536,752, U.S.Pat. No. 5,550,142, U.S. Pat. No. 5,604,260, U.S. Pat. No. 5,698,584,U.S. Pat. No. 5,710,140, WO 94/15932, U.S. Pat. No. 5,344,991, U.S. Pat.No. 5,134,142, U.S. Pat. No. 5,380,738, U.S. Pat. No. 5,393,790, U.S.Pat. No. 5,466,823, U.S. Pat. No. 5,633,272, and U.S. Pat. No.5,932,598, all of which are hereby incorporated by reference.

Inhibitors of COX-2 that are particularly useful in the instant methodof treatment are: 3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone;and5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine; ora pharmaceutically acceptable salt thereof.

Compounds that have been described as specific inhibitors of COX-2 andare therefore useful in the present invention include, but are notlimited to: parecoxib, CELEBREX® and BEXTRA® or a pharmaceuticallyacceptable salt thereof.

Other examples of angiogenesis inhibitors include, but are not limitedto, endostatin, ukrain, ranpirnase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]-methyl]-1H-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RPI4610, NX31838, sulfatedmannopentaose phosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

As used above, “integrin blockers” refers to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe α_(v)β₃ integrin, to compounds which selectively antagonize, inhibitor counteract binding of a physiological ligand to the αvβ5 integrin, tocompounds which antagonize, inhibit or counteract binding of aphysiological ligand to both the α_(v)β₃ integrin and the α_(v)β₅integrin, and to compounds which antagonize, inhibit or counteract theactivity of the particular integrin(s) expressed on capillaryendothelial cells. The term also refers to antagonists of the α_(v)β₆,α_(v)β₈, α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins. The term also refersto antagonists of any combination of α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈,α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins.

Some specific examples of tyrosine kinase inhibitors includeN-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one,SH268, genistein, imatinib (STI571), CEP2563,4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A,N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

Combinations with compounds other than anti-cancer compounds are alsoencompassed in the instant methods. For example, combinations of theinstantly claimed compounds with PPAR-γ (i.e., PPAR-gamma) agonists andPPAR-δ (i.e., PPAR-delta) agonists are useful in the treatment ofcertain malignancies. PPAR-γ and PPAR-δ are the nuclear peroxisomeproliferator-activated receptors γ and δ. The expression of PPAR-γ onendothelial cells and its involvement in angiogenesis has been reportedin the literature (see J. Cardiovasc. Pharmacol. 1998; 31:909-913; J.Biol. Chem. 1999; 274:9116-9121; Invest. Opthalmol. Vis. Sci. 2000;41:2309-2317). More recently, PPAR-γ agonists have been shown to inhibitthe angiogenic response to VEGF in vitro; both troglitazone androsiglitazone maleate inhibit the development of retinalneovascularization in mice. (Arch. Ophthamol. 2001; 119:709-717).Examples of PPAR-γ agonists and PPAR-γ/α agonists include, but are notlimited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone,rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate,GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544,NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926,2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionicacid (disclosed in U.S. Ser. No. 09/782,856), and2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylicacid (disclosed in U.S. Ser. No. 60/235,708 and 60/244,697).

Another embodiment of the instant invention is the use of the presentlydisclosed compounds in combination with gene therapy for the treatmentof cancer. For an overview of genetic strategies to treating cancer seeHall et al. (Am J Hum Genet. 61:785-789, 1997) and Kufe et al (CancerMedicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene therapycan be used to deliver any tumor suppressing gene. Examples of suchgenes include, but are not limited to, p53, which can be delivered viarecombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134,for example), Duc-4, NF-1, NF-2, RB, WT1, BRCA1, BRCA2, a uPA/uPARantagonist (“Adenovirus-Mediated Delivery of a uPA/uPAR AntagonistSuppresses Angiogenesis-Dependent Tumor Growth and Dissemination inMice,” Gene Therapy, August 1998; 5 (8):1105-13), and interferon gamma(J. Immunol. 2000; 164:217-222).

The compounds of the instant invention may also be administered incombination with an inhibitor of inherent multidrug resistance (MDR), inparticular MDR associated with high levels of expression of transporterproteins. Such MDR inhibitors include inhibitors of p-glycoprotein(P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833(valspodar).

A compound of the present invention may be employed in conjunction withanti-emetic agents to treat nausea or emesis, including acute, delayed,late-phase, and anticipatory emesis, which may result from the use of acompound of the present invention, alone or with radiation therapy. Forthe prevention or treatment of emesis, a compound of the presentinvention may be used in conjunction with other anti-emetic agents,especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists,such as ondansetron, granisetron, tropisetron, and zatisetron, GABABreceptor agonists, such as baclofen, a corticosteroid such as Decadron(dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten orothers such as disclosed in U.S. Pat. Nos. 2,789,118, 2,990,401,3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, anantidopaminergic, such as the phenothiazines (for exampleprochlorperazine, fluphenazine, thioridazine and mesoridazine),metoclopramide or dronabinol. In an embodiment, an anti-emesis agentselected from a neurokinin-1 receptor antagonist, a 5HT3 receptorantagonist and a corticosteroid is administered as an adjuvant for thetreatment or prevention of emesis that may result upon administration ofthe instant compounds.

Neurokinin-1 receptor antagonists of use in conjunction with thecompounds of the present invention are fully described, for example, inU.S. Pat. Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595,5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147; European PatentPublication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681, 0 517589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0545 478, 0 558 156, 0 577 394, 0 585 913, 0 590 152, 0 599 538, 0 610793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0699 674, 0707 006, 0708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733 632and 0 776 893; PCT International Patent Publication Nos. WO 90/05525,90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151, 92/15585,92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330, 93/00331,93/01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116, 93/10073,93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181, 93/23380,93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429, 94/03445,94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165, 94/10167,94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767, 94/15903,94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309, 95/02595,95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549, 95/11880,95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129, 95/19344,95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418, 95/30674,95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094, 96/07649,96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304, 96/29317,96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553, 97/01554,97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084, 97/19942 and97/21702; and in British Patent Publication Nos. 2 266 529, 2 268 931, 2269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293 169, and 2302 689. The preparation of such compounds is fully described in theaforementioned patents and publications, which are incorporated hereinby reference.

In an embodiment, the neurokinin-1 receptor antagonist for use inconjunction with the compounds of the present invention is selectedfrom:2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)-phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholine,or a pharmaceutically acceptable salt thereof, which is described inU.S. Pat. No. 5,719,147.

A compound of the instant invention may also be administered with anagent useful in the treatment of anemia. Such an anemia treatment agentis, for example, a continuous eythropoiesis receptor activator (such asepoetin alfa).

A compound of the instant invention may also be administered with anagent useful in the treatment of neutropenia. Such a neutropeniatreatment agent is, for example, a hematopoietic growth factor whichregulates the production and function of neutrophils such as a humangranulocyte colony stimulating factor, (G-CSF). Examples of a G-CSFinclude filgrastim.

A compound of the instant invention may also be administered with animmunologic-enhancing drug, such as levamisole, bacillusCalmette-Guerin, octreotide, isoprinosine and Zadaxin.

A compound of the instant invention may also be useful for treating orpreventing cancer, including bone cancer, in combination withbisphosphonates (understood to include bisphosphonates, diphosphonates,bisphosphonic acids and diphosphonic acids). Examples of bisphosphonatesinclude but are not limited to: etidronate (Didronel), pamidronate(Aredia), alendronate (Fosamax), risedronate (Actonel), zoledronate(Zometa), ibandronate (Boniva), incadronate or cimadronate, clodronate,EB-1053, minodronate, neridronate, piridronate and tiludronate includingany and all pharmaceutically acceptable salts, derivatives, hydrates andmixtures thereof.

A compound of the instant invention may also be useful for treating orpreventing breast cancer in combination with aromatase inhibitors.Examples of aromatase inhibitors include but are not limited toanastrozole, letrozole and exemestane.

A compound of the instant invention may also be useful for treating orpreventing cancer in combination with siRNA therapeutics.

A compound of the instant invention may also be useful for treating orpreventing cancer in combination with compounds which induce terminaldifferentiation of the neoplastic cells. Suitable differentiation agentsinclude the compounds disclosed in any one or more of the followingreferences, the contents of which are incorporated by reference herein.

a) Polar compounds (Marks et al (1987); Friend, C., Scher, W., Holland,J. W., and Sato, T. (1971) Proc. Natl. Acad. Sci. (USA) 68: 378-382;Tanaka, M., Levy, J., Terada, M., Breslow, R., Rifkind, R. A., andMarks, P. A. (1975) Proc. Natl. Acad. Sci. (USA) 72: 1003-1006; Reuben,R. C., Wife, R. L., Breslow, R., Rifkind, R. A., and Marks, P. A. (1976)Proc. Natl. Acad. Sci. (USA) 73: 862-866);

b) Derivatives of vitamin D and retinoic acid (Abe, E., Miyaura, C.,Sakagami, H., Takeda, M., Konno, K., Yamazaki, T., Yoshika, S., andSuda, T. (1981) Proc. Natl. Acad. Sci. (USA) 78: 4990-4994; Schwartz, E.L., Snoddy, J. R., Kreutter, D., Rasmussen, H., and Sartorelli, A. C.(1983) Proc. Am. Assoc. Cancer Res. 24: 18; Tanenaga, K., Hozumi, M.,and Sakagami, Y. (1980) Cancer Res. 40: 914-919);

c) Steroid hormones (Lotem, J. and Sachs, L. (1975) Int. J. Cancer 15:731-740);

d) Growth factors (Sachs, L. (1978) Nature (Lond.) 274: 535, Metcalf, D.(1985) Science, 229: 16-22);

e) Proteases (Scher, W., Scher, B. M., and Waxman, S. (1983) Exp.Hematol. 11: 490-498; Scher, W., Scher, B. M., and Waxman, S. (1982)Biochem. & Biophys. Res. Comm. 109: 348-354);

f) Tumor promoters (Huberman, E. and Callaham, M. F. (1979) Proc. Natl.Acad. Sci. (USA) 76: 1293-1297; Lottem, J. and Sachs, L. (1979) Proc.Natl. Acad. Sci. (USA) 76: 5158-5162); and

g) inhibitors of DNA or RNA synthesis (Schwartz, E. L. and Sartorelli,A. C. (1982) Cancer Res. 42: 2651-2655, Terada, M., Epner, E., Nudel,U., Salmon, J., Fibach, E., Rifkind, R. A., and Marks, P. A. (1978)Proc. Natl. Acad. Sci. (USA) 75: 2795-2799; Morin, M. J. and Sartorelli,A. C. (1984) Cancer Res 44: 2807-2812; Schwartz, E. L., Brown, B. J.,Nierenberg, M., Marsh, J. C., and Sartorelli, A. C. (1983) Cancer Res.43: 2725-2730; Sugano, H., Furusawa, M., Kawaguchi, T., and Ikawa, Y.(1973) Bibl. Hematol. 39: 943-954; Ebert, P. S., Wars, I., and Buell, D.N. (1976) Cancer Res. 36:1809-1813; Hayashi, M., Okabe, J., and Hozumi,M. (1979) Gann 70: 235-238).

A compound of the instant invention may also be useful for treating orpreventing cancer in combination with γ-secretase inhibitors.

Also included in the scope of the claims is a method of treating cancerthat comprises administering a therapeutically effective amount of acompound of the instant invention in combination with radiation therapyand/or in combination with a second compound selected from: an estrogenreceptor modulator, an androgen receptor modulator, a retinoid receptormodulator, a cytotoxiccytostatic agent, an antiproliferative agent, aprenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, anHIV protease inhibitor, a reverse transcriptase inhibitor, anangiogenesis inhibitor, PPAR-γ agonists, PPAR-δ agonists, an inhibitorof inherent multidrug resistance, an anti-emetic agent, an agent usefulin the treatment of anemia, an agent useful in the treatment ofneutropenia, an immunologic-enhancing drug, an inhibitor of cellproliferation and survival signaling, a bisphosphonate, an aromataseinhibitor, an siRNA therapeutic, γ-secretase inhibitors, agents thatinterfere with receptor tyrosine kinases (RTKs) and an agent thatinterferes with a cell cycle checkpoint.

The compounds of the instant invention are useful in combination withthe following therapeutic agents: abarelix (Plenaxis Depot®);aldesleukin (Prokine®); Aldesleukin (Proleukin®); Alemtuzumabb(Campath®); alitretinoin (Panretin®); allopurinol (Zyloprim®);altretamine (Hexylen®); amifostine (Ethyol®); anastrozole (Arimidex®);arsenic trioxide (Trisenox®); asparaginase (Elspar®); azacitidine(Vidaza®); bevacuzimab (Avastin®); bexarotene capsules (Targretin®);bexarotene gel (Targretin®); bleomycin (Blenoxane®); bortezomib(Velcade®); busulfan intravenous (Busulfex®); busulfan oral (Myleran®);calusterone (Methosarb®); capecitabine (Xeloda®); carboplatin(Paraplatin®); carmustine (BCNU®, BiCNU®); carmustine (Gliadel®);carmustine with Polifeprosan 20 Implant (Gliadel Wafer®); celecoxib(Celebrex®); cetuximab (Erbitux®); chlorambucil (Leukeran®); cisplatin(Platinol®); cladribine (Leustatin®, 2-CdA®); clofarabine (Clolar®);cyclophosphamide (Cytoxan®, Neosar®); cyclophosphamide (CytoxanInjection®); cyclophosphamide (Cytoxan Tablet®); cytarabine(Cytosar-U®); cytarabine liposomal (DepoCyt®); dacarbazine (DTIC-Dome®);dactinomycin, actinomycin D (Cosmegen®); Darbepoetin alfa (Aranesp®);daunorubicin liposomal (DanuoXome®); daunorubicin, daunomycin(Daunorubicin®); daunorubicin, daunomycin (Cerubidine®); Denileukindiftitox (Ontak®); dexrazoxane (Zinecard®); docetaxel (Taxotere®);doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®, Rubex®);doxorubicin (Adriamycin PFS Injection®); doxorubicin liposomal (Doxil®);dromostanolone propionate (Dromostanolon®); dromostanolone propionate(masterone Injection®); Elliott's B Solution (Elliott's B Solution®);epirubicin (Ellence®); Epoetin alfa (Epogen®); erlotinib (Tarceva®);estramustine (Emcyt®); etoposide phosphate (Etopophos®); etoposide,VP-16 (Vepesid®); exemestane (Aromasin®); Filgrastim (Neupogen®);floxuridine (intraarterial) (FUDR®); fludarabine (Fludara®);fluorouracil, 5-FU (Adrucil®); fulvestrant (Faslodex®); gefitinib(Iressa®); gemcitabine (Gemzar®); gemtuzumab ozogamicin (Mylotarg®);goserelin acetate (Zoladex Implant®); goserelin acetate (Zoladex®);histrelin acetate (Histrelin Implant®); hydroxyurea (Hydrea®);Ibritumomab Tiuxetan (Zevalin®); idarubicin (Idamycin®); ifosfamide(IFEX®); imatinib mesylate (Gleevec®); interferon alfa 2a (Roferon A®);Interferon alfa-2b (Intron A®); irinotecan (Camptosar®); lenalidomide(Revlimid®); letrozole (Femara®); leucovorin (Wellcovorin®,Leucovorin®); Leuprolide Acetate (Eligard®); levamisole (Ergamisol®);lomustine, CCNU (CeeBU®); meclorethamine, nitrogen mustard (Mustargen®);megestrol acetate (Megace®); melphalan, L-PAM (Alkeran®);mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®); mesna (MesnexTabs®); methotrexate (Methotrexate®); methoxsalen (Uvadex®); mitomycin C(Mutamycin®); mitotane (Lysodren®); mitoxantrone (Novantrone®);nandrolone phenpropionate (Durabolin-50®); nelarabine (Arranon®);Nofetumomab (Verluma®); Oprelvekin (Neumega®); oxaliplatin (Eloxatin®);paclitaxel (Paxene®); paclitaxel (Taxol®); paclitaxel protein-boundparticles (Abraxane®); palifermin (Kepivance®); pamidronate (Aredia®);pegademase (Adagen (Pegademase Bovine)®); pegaspargase (Oncaspar®);Pegfilgrastim (Neulasta®); pemetrexed disodium (Alimta®); pentostatin(Nipent®); pipobroman (Vercyte®); plicamycin, mithramycin (Mithracin®);porfimer sodium (Photofrin®); procarbazine (Matulane®); quinacrine(Atabrine®); Rasburicase (Elitek®); Rituximab (Rituxan®); sargramostim(Leukine®); Sargramostim (Prokine®); sorafenib (Nexavar®); streptozocin(Zanosar®); sunitinib maleate (Sutent®); talc (Sclerosol®); tamoxifen(Nolvadex®); temozolomide (Temodar®); teniposide, VM-26 (Vumon®);testolactone (Teslac®); thioguanine, 6-TG (Thioguanine®); thiotepa(Thioplex®); topotecan (Hycamtin®); toremifene (Fareston®); Tositumomab(Bexxar®); Tositumomab/I-131 tositumomab (Bexxar®); Trastuzumab(Herceptin®); tretinoin, ATRA (Vesanoid®); Uracil Mustard (UracilMustard Capsules®); valrubicin (Valstar); vinblastine (Velban®));vincristine (Oncovin®); vinorelbine (Navelbine®); vinorelbine(Navelbine®); zoledronate (Zometa®); and zoledronic acid (Zometa®).

The use of all of these approaches in combination with the compounds ofthe instant invention, as described herein, are within the scope of thepresent invention.

Dosages and Dosing Schedules

The dosage regimen utilizing the compounds of the present invention canbe selected in accordance with a variety of factors including type,species, age, weight, sex and the type of cancer being treated; theseverity (i.e., stage) of the disease to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound or salt thereof employed. An ordinarily skilledphysician or veterinarian can readily determine and prescribe theeffective amount of the drug required to treat, for example, to prevent,inhibit (fully or partially) or arrest the progress of the disease.

For oral administration, suitable daily dosages are for example betweenabout 5-4000 mg/m² administered orally once-daily, twice-daily or threetimes-daily, continuous (every day) or intermittently (e.g., 3-5 days aweek). For example, when used to treat the desired disease, the dose ofthe compounds of the instant invention can range between about 2 mg toabout 2000 mg per day.

The compound of the instant invention may be administered once daily(QD), or divided into multiple daily doses such as twice daily (BID),and three times daily (TID). For administration once a day, a suitablyprepared medicament would therefore contain all of the needed dailydose. For administration twice a day, a suitably prepared medicamentwould therefore contain half of the needed daily dose. Foradministration three times a day, a suitably prepared medicament wouldtherefore contain one third of the needed daily dose.

In addition, the administration can be continuous, i.e., every day, orintermittently. The terms “intermittent” or “intermittently” as usedherein means stopping and starting at either regular or irregularintervals. For example, intermittent administration of an HDAC inhibitormay be administration one to six days per week or it may meanadministration in cycles (e.g., daily administration for two to eightconsecutive weeks, then a rest period with no administration for up toone week) or it may mean administration on alternate days.

Typically, an intravenous formulation may be prepared which contains aconcentration of the compounds of the instant invention of between about1.0 mg/mL to about 10 mg/mL. In one example, a sufficient volume ofintravenous formulation can be administered to a patient in a day suchthat the total dose for the day is between about 10 and about 1500mg/m².

Subcutaneous formulations, preferably prepared according to procedureswell known in the art at a pH in the range between about 5 and about 12,also include suitable buffers and isotonicity agents, as describedbelow. They can be formulated to deliver a daily dose of HDAC inhibitorin one or more daily subcutaneous administrations, e.g., one, two orthree times each day.

The compounds can also be administered in intranasal form via topicaluse of suitable intranasal vehicles, or via transdermal routes, usingthose forms of transdermal skin patches well known to those of ordinaryskill in that art. To be administered in the form of a transdermaldelivery system, the dosage administration will, or course, becontinuous rather than intermittent throughout the dosage regime.

It should be apparent to a person skilled in the art that the variousmodes of administration, dosages and dosing schedules described hereinmerely set forth specific embodiments and should not be construed aslimiting the broad scope of the invention. Any permutations, variationsand combinations of the dosages and dosing schedules are included withinthe scope of the present invention.

The term “administration” and variants thereof (e.g., “administering” acompound) in reference to a compound of the invention means introducingthe compound or a prodrug of the compound into the system of the animalin need of treatment. When a compound of the invention or prodrugthereof is provided in combination with one or more other active agents(e.g., a cytotoxic agent, etc.), “administration” and its variants areeach understood to include concurrent and sequential introduction of thecompound or prodrug thereof and other agents.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

The term “therapeutically effective amount” as used herein means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician.

Pharmaceutical Compositions

The compounds of the invention, and derivatives, fragments, analogs,homologs pharmaceutically acceptable salts or hydrate thereof, can beincorporated into pharmaceutical compositions suitable for oraladministration, together with a pharmaceutically acceptable carrier orexcipient. Such compositions typically comprise a therapeuticallyeffective amount of any of the compounds above, and a pharmaceuticallyacceptable carrier. In one embodiment, the effective amount is an amounteffective to selectively induce terminal differentiation of suitableneoplastic cells and less than an amount which causes toxicity in apatient.

Any inert excipient that is commonly used as a carrier or diluent may beused in the formulations of the present invention, such as for example,a gum, a starch, a sugar, a cellulosic material, an acrylate, ormixtures thereof. A preferred diluent is microcrystalline cellulose. Thecompositions may further comprise a disintegrating agent (e.g.,croscarmellose sodium) and a lubricant (e.g., magnesium stearate), andin addition may comprise one or more additives selected from a binder, abuffer, a protease inhibitor, a surfactant, a solubilizing agent, aplasticizer, an emulsifier, a stabilizing agent, a viscosity increasingagent, a sweetener, a film forming agent, or any combination thereof.Furthermore, the compositions of the present invention may be in theform of controlled release or immediate release formulations.

In one embodiment, the pharmaceutical compositions are administeredorally, and are thus formulated in a form suitable for oraladministration, i.e., as a solid or a liquid preparation. Suitable solidoral formulations include tablets, capsules, pills, granules, pelletsand the like. Suitable liquid oral formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In oneembodiment of the present invention, the composition is formulated in acapsule. In accordance with this embodiment, the compositions of thepresent invention comprise in addition to a compound of the instantinvention and the inert carrier or diluent, a hard gelatin capsule.

As used herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration, such as sterilepyrogen-free water. Suitable carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, a standard referencetext in the field, which is incorporated herein by reference. Preferredexamples of such carriers or diluents include, but are not limited to,water, saline, finger's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

Solid carriers/diluents include, but are not limited to, a gum, a starch(e.g., corn starch, pregelatinized starch), a sugar (e.g., lactose,mannitol, sucrose, dextrose), a cellulosic material (e.g.,microcrystalline cellulose), an acrylate (e.g., polymethylacrylate),calcium carbonate, magnesium oxide, talc, or mixtures thereof.

For liquid formulations, pharmaceutically acceptable carriers may beaqueous or non-aqueous solutions, suspensions, emulsions or oils.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Examples of oils arethose of petroleum, animal, vegetable, or synthetic origin, for example,peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, andfish-liver oil. Solutions or suspensions can also include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide.

In addition, the compositions may further comprise binders (e.g.,acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),disintegrating agents (e.g., cornstarch, potato starch, alginic acid,silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodiumstarch glycolate, Primogel), buffers (e.g., tris-HCl, acetate,phosphate) of various pH and ionic strength, additives such as albuminor gelatin to prevent absorption to surfaces, detergents (e.g., Tween20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors,surfactants (e.g., sodium lauryl sulfate), permeation enhancers,solubilizing agents (e.g., glycerol, polyethylene glycerol), a glidant(e.g., colloidal silicon dioxide), anti-oxidants (e.g., ascorbic acid,sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g.,hydroxypropyl cellulose, hydroxypropylmethyl cellulose), viscosityincreasing agents (e.g., carbomer, colloidal silicon dioxide, ethylcellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citricacid), flavoring agents (e.g., peppermint, methyl salicylate, or orangeflavoring), preservatives (e.g., Thimerosal, benzyl alcohol, parabens),lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol,sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide),plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers(e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate),polymer coatings (e.g., poloxamers or poloxamines), coating and filmforming agents (e.g., ethyl cellulose, acrylates, polymethacrylates)and/or adjuvants.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The compounds of the present invention may be administered intravenouslyon the first day of treatment, with oral administration on the secondday and all consecutive days thereafter.

The compounds of the present invention may be administered for thepurpose of preventing disease progression or stabilizing tumor growth.

The preparation of pharmaceutical compositions that contain an activecomponent is well understood in the art, for example, by mixing,granulating, or tablet-forming processes. The active therapeuticingredient is often mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient. For oraladministration, the active agents are mixed with additives customary forthis purpose, such as vehicles, stabilizers, or inert diluents, andconverted by customary methods into suitable forms for administration,such as tablets, coated tablets, hard or soft gelatin capsules, aqueous,alcoholic or oily solutions and the like as detailed above.

The amount of the compound administered to the patient is less than anamount that would cause toxicity in the patient. In the certainembodiments, the amount of the compound that is administered to thepatient is less than the amount that causes a concentration of thecompound in the patient's plasma to equal or exceed the toxic level ofthe compound. In one embodiment, the concentration of the compound inthe patient's plasma is maintained at about 10 nM. In anotherembodiment, the concentration of the compound in the patient's plasma ismaintained at about 25 nM. In another embodiment, the concentration ofthe compound in the patient's plasma is maintained at about 50 nM. Inanother embodiment, the concentration of the compound in the patient'splasma is maintained at about 100 nM. In another embodiment, theconcentration of the compound in the patient's plasma is maintained atabout 500 nM. In another embodiment, the concentration of the compoundin the patient's plasma is maintained at about 1000 nM. In anotherembodiment, the concentration of the compound in the patient's plasma ismaintained at about 2500 nM. In another embodiment, the concentration ofthe compound in the patient's plasma is maintained at about 5000 nM. Theoptimal amount of the compound that should be administered to thepatient in the practice of the present invention will depend on theparticular compound used and the type of cancer being treated.

The instant invention also includes a pharmaceutical composition usefulfor treating or preventing cancer that comprises a therapeuticallyeffective amount of a compound of Formula I and a second compoundselected from: an estrogen receptor modulator, an androgen receptormodulator, a retinoid receptor modulator, a cytotoxic/cytostatic agent,an antiproliferative agent, a prenyl-protein transferase inhibitor, anHMG-CoA reductase inhibitor, an HIV protease inhibitor, a reversetranscriptase inhibitor, an angiogenesis inhibitor, a PPAR-γ agonist, aPPAR-δ agonist, an inhibitor of cell proliferation and survivalsignaling, a bisphosphonate, an aromatase inhibitor, an siRNAtherapeutic, γ-secretase inhibitors, agents that interfere with receptortyrosine kinases (RTKs) and an agent that interferes with a cell cyclecheckpoint.

In Vitro Methods:

The present invention also provides methods of using the compounds ofthe present invention for inducing terminal differentiation, cell growtharrest and/or apoptosis of neoplastic cells thereby inhibiting theproliferation of such cells. The methods can be practiced in vivo or invitro.

In one embodiment, the present invention provides in vitro methods forselectively inducing terminal differentiation, cell growth arrest and/orapoptosis of neoplastic cells, thereby inhibiting proliferation of suchcells, by contacting the cells with an effective amount of any one ormore of the compounds of the instant invention described herein.

In a particular embodiment, the present invention relates to an in vitromethod of selectively inducing terminal differentiation of neoplasticcells and thereby inhibiting proliferation of such cells. The methodcomprises contacting the cells under suitable conditions with aneffective amount of one or more of the compounds of the instantinvention described herein.

In another embodiment, the invention relates to an in vitro method ofselectively inducing cell growth arrest of neoplastic cells and therebyinhibiting proliferation of such cells. The method comprises contactingthe cells under suitable conditions with an effective amount of one ormore of the compounds of the instant invention described herein.

In another embodiment, the invention relates to an in vitro method ofselectively inducing apoptosis of neoplastic cells and therebyinhibiting proliferation of such cells. The method comprises contactingthe cells under suitable conditions with an effective amount of one ormore of the compounds of the instant invention described herein.

In another embodiment, the invention relates to an in vitro method ofinducing terminal differentiation of tumor cells in a tumor comprisingcontacting the cells with an effective amount of any one or more of thecompounds of the instant invention described herein.

In one embodiment, the methods of selectively inducing terminaldifferentiation, cell growth arrest and/or apoptosis of neoplasticcells, and of inhibiting HDAC will comprise contacting the cells invivo, i.e., by administering the compounds to a subject harboringneoplastic cells or tumor cells in need of treatment.

Thus, the present invention provides in vivo methods for selectivelyinducing terminal differentiation, cell growth arrest and/or apoptosisof neoplastic cells in a subject, thereby inhibiting proliferation ofsuch cells in the subject, by administering to the subject an effectiveamount of any one or more of the compounds of the instant inventiondescribed herein.

In a particular embodiment, the present invention relates to a method ofselectively inducing terminal differentiation of neoplastic cells andthereby inhibiting proliferation of such cells in a subject. The methodcomprises administering to the subject an effective amount of one ormore of the compounds of the instant invention described herein.

In another embodiment, the invention relates to a method of selectivelyinducing cell growth arrest of neoplastic cells and thereby inhibitingproliferation of such cells in a subject. The method comprisesadministering to the subject an effective amount of one or more of thecompounds of the instant invention described herein.

In another embodiment, the invention relates to a method of selectivelyinducing apoptosis of neoplastic cells and thereby inhibitingproliferation of such cells in a subject. The method comprisesadministering to the subject an effective amount of one or more of thecompounds of the instant invention described herein.

In another embodiment, the invention relates to a method of treating apatient having a tumor characterized by proliferation of neoplasticcells. The method comprises administering to the patient one or more ofthe compounds of the instant invention described herein. The amount ofcompound is effective to selectively induce terminal differentiation,induce cell growth arrest and/or induce apoptosis of such neoplasticcells and thereby inhibit their proliferation.

The invention is illustrated in the following generic schemes and theexamples that follow. This section is set forth to aid in anunderstanding of the invention but is not intended to, and should not beconstrued to limit in any way the invention as set forth in the claimswhich follow thereafter.

Synthesis of Common Intermediates

tert-Butyl (2-Aminophenyl)carbamate (A). Intermediate A was prepared bythe method described by Seto, C. T.; Mathias, J. P.; Whitesides, G. M.Molecular self-assembly through hydrogen bonding: aggregation of fivemolecules to form a discrete supramolecular structure. J. Am. Chem. Soc.1993, 115, 1321.

tert-Butyl (2-{[(6-Chloropyridin-3-yl)carbonyl]amino}phenyl)carbamate(B). To a solution of t-butyl (2-aminophenyl)carbamate A (10 g, 48.0mmol) in CH₂Cl₂ (200 mL) was added 6-chloronicotinoyl chloride (8.5 g,48.0 mmol). The reaction mixture was concentrated after 2 hours ofstirring at room temperature and purified by flash chromatography(10-75% EtOAc/hexanes) to give the Boc-protected nicotinamide Bconfirmed by MS (ESI+): cal'd [M+Na]⁺ 370.1, obs'd 370.1.

tert-Butyl (3-Aminobiphenyl-4-yl)carbamate (C). Intermediate C wasprepared from tert-butyl (4-bromo-2-nitrophenyl)carbamate as describedin a published procedure; see Adam, G.; Alanine, A.; Goetschi, E.;Mutel, V.; Woltering, T. J. Preparation of benzodiazepine derivatives asmetabotropic glutamate receptor antagonists. PCT Int. Appl. (2001) WO2001029012 A2.

A mixture of N-Boc 4-bromo-2-nitroaniline (39.0 g, 123 mmol),phenylboronic acid (16.5 g, 135 mmol) and K₂CO₃ (34.1 g, 247 mmol) in350 mL of dioxane and 150 mL of water was degassed by bubbling nitrogenthrough the mixture for 30 min. Next, Pd(PPh₃)₄ was added (4.32 g, 3.7mmol) and the orange mixture was warmed to 78° C. for 18 h. Cooled andpartitioned between ether (1500 mL) and water (400 mL). Filtered mixturethrough a pad of Celite (w/ether washes). Organic layer was separated,washed with brine, dried (MgSO₄) and concentrated to afford 44.1 g ofreddish-orange solid. Recrystallization from EtOAc-hexanes (ca. 50mL+1100 mL, respectively) afforded the bright orange solid N-Boc4-phenyl-2-nitroaniline: MS (EI) [M+Na]⁺ cal'd 337.2, obs'd 337.2.

A solution of nitro compound (16.5 g, 52.5 mmol) in 400 mL of EtOAcevacuated and refilled with nitrogen (2×). Added 10% Pd/C (1.60 g), thenevacuated and refilled with hydrogen (3×). Stirred under atmosphere ofhydrogen overnight. Mixture was filtered through a pad of Celite(w/EtOAc, then CH₂Cl₂ washes) and concentrated to a pale orange solid.Stirred and warmed with ca. 800 mL of hexanes, then cooled and collectedproduct (w/cold hexane washes). Dissolved resulting solid in CH₂Cl₂ andconcentrated to provide the off-white solid N—BOC(3-aminobiphenyl-4-yl)amine C: ¹H NMR (600 MHz, CDCl₃) δ 7.51 (d, J=3.2Hz, 2H), 7.38 (t, J=5.6 Hz, 2H), 7.31 (m, 2H), 7.22 (s, 1H), 7.12 (dd,J=8.2, 2.1 Hz, 1H), 6.45 (br s, 1H), 1.51 (s, 9H); MS (EI) [M+Na]⁺ cal'd285.1, obs'd 285.1.

tert-Butyl(3-{[(6-Chloropyridin-3-yl)carbonyl]amino}biphenyl-4-yl)carbamate (D).To a solution of t-butyl (3-aminobiphenyl-4-yl)carbamate C (2.06 g, 7.25mmol) in pyridine (10 mL) was added 6-chloronicotinyl chloride (1.30 g,7.39 mmol). After 4 hours of stirring at room temperature, the reactionmixture was filtered and the solvent concentrated. Formation oftert-butyl(3-{[(6-chloropyridin-3-yl)carbonyl]amino}biphenyl-4-yl)carbamate(D) was confirmed by ¹H NMR (600 MHz, CD₃OD): δ 10.84 (s, 1H), 9.79 (s,1H), 9.60 (s, 1H), 9.19-9.16 (m, 1H), 8.59 (s, 1H), 8.57-8.55 (m, 2H),8.43-8.40 (m, 2H), 8.34-8.30 (m, 1H), 8.25-8.21 (m, 2H), 8.16-8.12 (m,1H), 2.22 (s, 9H).

tert-Butyl[2-Amino-4-(2-thienyl)phenyl]carbamate (E). Intermediate E wasprepared from tert-butyl (4-bromo-2-nitrophenyl)carbamate as describedin a published procedure; see Adam, G.; Alanine, A.; Goetschi, E.;Mutel, V.; Woltering, T. J. Preparation of benzodiazepine derivatives asmetabotropic glutamate receptor antagonists. PCT Int. Appl. (2001) WO2001029012 A2.

A mixture of tert-butyl (4-bromo-2-nitrophenyl)carbamate (19.4 g, 61.2mmol), thiophene-2-boronic acid (9.94 g, 77.7 mmol) and K₂CO₃ (22.2 g,160 mmol) in 60 mL of dioxane and 60 mL of water was degassed bybubbling nitrogen through the mixture for 30 min. Next, Pd[PPh₃]₄ (5.25g, 4.53 mmol) was added and the heterogeneous mixture was warmed toreflux for 20 h. The mixture was cooled and diluted with ethyl acetate,washed with water and brine, dried (MgSO₄), and concentrated. Theresulting solid was dissolved in diethylether (500 mL) and filteredthrough a pad of silica. The solvents were removed under reducedpressure to yield the yellow-brown solid. This solid was taken directlyto the next step without further purification: ¹H NMR (600 MHz, CDCl₃):δ 9.65 (s, 9H), 8.58 (d, J=8.8 Hz, 1H), 8.39 (d, J=2.1 Hz, 1H), 7.81(dd, J=8.8, 1.8 Hz, 1H), 7.32 (m, 2H), 7.09 (dd, J=5.3, 3.8 Hz, 1H),1.54 (s, 9H); MS (ESI+): cal'd [M+Na]⁺ 343.1, obs'd 343.1.

A solution of N—BOC 2-nitro-4-(2-thienyl)aniline (18.0 g) in 350 mL ofEtOAc was evacuated and refilled with nitrogen (2×). To the solution wasadded 10% Pd/C (4.46 g), and the reaction mixture was evacuated andrefilled with hydrogen (2×). The black reaction mixture was stirredunder an atmosphere of hydrogen overnight. The mixture was filteredthrough a pad of celite (with EtOAc then CH₂Cl₂ washes) and concentratedto provide a brownish-white solid. The solid was triturated with etherand filtered to provide the off-whitetert-butyl[2-amino-4-(2-thienyl)phenyl]carbamate (E): ¹H NMR (600 MHz,DMSO-d₆) δ 8.31 (br, 1H), 7.41 (dd, J=5.0, 0.9 Hz, 1H), 7.26 (dd, J=3.5,1.2 Hz, 1H), 7.23 (br d, J=8.5 Hz, 1H), 7.05 (dd, J=5.0, 3.5 Hz, 1H),6.94 (d, J=2.1 Hz, 1H), 6.81 (dd, J=8.2, 2.1 Hz, 1H), 4.98 (s, 2H), 1.43(s, 9H); MS (ESI+): cal'd [M+Na]⁺ 291.1, obs'd 291.1.

tert-Butyl[2-{[(6-Chloropyridin-3-yl)carbonyl]amino}-4-(2-thienyl)phenyl]-carbamate(F). A mixture of tert-butyl[2-amino-4-(2-thienyl)phenyl]carbamate (E)(600 mg, 2.07 mmol) and 6-chloronicotinyl chloride (380 mg, 2.16 mmol)in 5 mL of pyridine was stirred overnight, poured into EtOAc and washedwith saturated NaHCO₃, dried (Na₂SO₄) and concentrated giving theBOC-protected chloronicotinamide (F): ¹H NMR (600 MHz, CD₃OD): δ 8.95(d, J=2.3 Hz, 1H), 8.35 (dd, J=8.2 Hz, 2.3 Hz, 1H), 7.85 (br s, 1H),7.62 (d, J=8.5 Hz, 1H), 7.55-7.51 (m, 2H), 7.37-7.35 (m, 2H), 7.07 (dd,J=5.0 Hz, 3.5 Hz, 1H), 4.59 (s, 1H), 1.49 (s, 9H); MS (ESI+): cal'd[M+Na]⁺ 452.1, obs'd 452.1.

tert-Butyl[2-Amino-4-(3-thienyl)phenyl]carbamate (G) andtert-Butyl[2-{[(6-Chloropyridin-3-yl)carbonyl]amino}-4-(3-thienyl)phenyl]-carbamate(H). Intermediates G and H were prepared from tert-butyl(4-bromo-2-nitrophenyl)carbamate using the methods employed for thepreparation of intermediates E and F. Intermediate G: MS (ESI+): cal'd[M+Na]⁺ 291.1, obs'd 291.1. Intermediate H: MS (ESI+): cal'd [M+Na]⁺452.1, obs'd 452.1.

Synthesis of Spirocyclic Precursors

-   AA, 1,2-Dihydrospiro[indole-3,4′-piperidine]    For synthetic procedure, see: Xie, J.-S., Huang, C. Q., Fang, Y.-Y.    and Zhu, Y.-F. A Convenient Synthesis of    1′-H-Spiro-(Indoline-3,4′-Piperidine) and its Derivatives.    Tetrahedron 2004, 60, 4875.-   BB, 1-Acetyl-1,2-dihydrospiro[indole-3,4′-piperidine]-   CC,    1-[(4-Methylphenyl)sulfonyl]-1,2-dihydrospiro[indole-3,4′-piperidine]-   QQ, Spiro[1-benzofuran-3,4′-piperidine]-   RR, 1-(Methylsulfonyl)-1,2-dihydrospiro[indole-3,4′-piperidine]    For synthetic procedure, see: Mills, S. G.; Springer, M. S.;    MacCoss, M. Preparation of Spiro-substituted Azacycles as Modulators    of Chemokine Receptor Activity. PCT Int. Appl. (1998), WO 9825605    A1.-   DD, tert-Butyl    1H-Spiro[isoquinoline-4,4′-piperidine]-2(3H)-carboxylate-   EE, tert-Butyl    2,3-Dihydro-1H,1′H-spiro[isoquinoline-4,4′-piperidine]-1′-carboxylate-   JJ, Spiro[chromene-2,4′-piperidin]-4(3H)-one-   KK, Spiro[isochromene-3,4′-piperidin]-1(4H)-one-   MM, 6-Methoxy-4-phenylspiro[isochromene-3,4′-piperidin]-1(4H)-one    For synthetic procedure, see: Chen, M.-H.; Johnston, D. B. R.;    Nargund, R. P.; Patchett, A. A.; Tata, J. R.; Yang, L. Spiro    Piperidines and Homologs Which Promote Release of Growth Hormone.    PCT Int. Appl. (1994), WO 9413696 A1.-   FF, Spiro[indeno[2,1-b]pyridine-9,4′-piperidine]    For synthetic procedure, see: Evans, B. E.; Gilbert, K. F.;    Hoffman, J. M.; Rittle, K. E. Preparation of Novel Spirotricyclic    Substituted Azacycloalkanes as α1a Adrenoceptor Antagonists. Brit.    UK Pat. Appl. (2001), GB 2355457 A1.-   GG,    1,3-Diethyl-4,5-dihydro-1H-spiro[cyclopenta[c]pyrazole-6,4′-piperidine]    For synthetic procedure, see: Finke, P. E.; Loebach, J. L.;    Parker, K. A.; Plummer, C. W.; Mills, S. G. Preparation of Amino    Acid N-Cyclopentyl Modulators of Chemokine Receptor Activity. U.S.    Pat. Appl. Publ. (2005), US 2005070609 A1.-   HH, Spiro[isoindole-1,4′-piperidin]-3(2H)-one    For synthetic procedure, see: Finke, P. E.; Meurer, L. C.; Oates,    B.; Shah, S. K.; Loebach, J. L.; Mills, S. G.; MacCoss, M.;    Castonguay, L.; Malkowitz, L.; Springer, M. S.; Gould, S. L.;    DeMartino, J. A. Antagonists of the Human CCR5 Receptor as    Anti-HIV-1 Agents. Part 3: A Proposed Pharmacophore Model for    1-[N-(Methyl)-N-(phenylsulfonyl)amino]-2-(phenyl)-4-[4-(substituted)piperidin-1-yl]butanes.    Bio. Med. Chem. Lett. 2001, 11, 2469.-   II,    (3R)—N-methyl-2,3-dihydrospiro[indene-1,4′-piperidine]-3-carboxamide    For synthetic procedure, see: Chen, M. H.; Lu, Z.; Nargund, R.;    Patchett, A. A.; Tata, J. R.; Wu, M. T.; Yang, L. Preparation of    Amino Acid Piperidine, Pyrrolidine, and Hexahydro-1H-azepine    Derivatives for Promoting Release of Growth Hormone. PCT Int. Appl.    (1998), WO 9825897 A1.-   LL, Ethyl 2,3-Dihydrospiro[indene-1,4′-piperidin]-3-ylcarbamate    For synthetic procedure, see: Chen, M.-H.; Johnston, D. B. R.;    Nargund, R. P.; Patchett, A. A.; Tata, J. R.; Yang, L. Preparation    of N-(α-Aminoalkanoyl)spiropiperidines as Growth Hormone Release    Promoters. U.S. (1996), U.S. Pat. No. 5,578,593 A.-   NN,    1-Methyl-5-phenylspiro[1,4-benzodiazepine-3,4′-piperidin]-2(1H)-one    For synthetic procedure, see: Boltze, K. H.; Etschenberg, E.;    Traber, J.; Buesgen, H. Spirobenzodiazepines. Ger. Offen. (1986), DE    3431195 A1.-   OO,    1-Isopropyl-6-methoxy-5-methylspiro[indole-3,4′-piperidin]-2(1H)-one-   PP,    5-(Benzylamino)-1-isopropylspiro[indole-3,4′-piperidin]-2(1H)-one    For synthetic procedure, see: Irie, O.; Iwasaki, G.; Masuya, K.;    Miyake, T.; Teno, N. Preparation of Spiro-Substituted    Pyrrolopyrimidines as Inhibitors of Cathepsin S and/or Cathepsin K.    PCT Int. Appl. (2004), WO 2004076455 A1.

General Procedures

Compounds 1-20 were prepared via the addition of a spirocycle AA-RR to achloronicotinamide such as B, D, F or H. The Boc protective group wasthen removed by treatment with TFA to yield the desired compound.Several examples follow.

Example 1 SYNTHESIS OFN-(2-AMINOPHENYL)-6-{1-[(4-METHYLPHENYL)SULFONYL]-1,2-DIHYDRO-1′H-SPIRO[INDOLE-3,4′-PIPERIDIN]-1′-YL}NICOTINAMIDE

N-(2-Aminophenyl)-6-{1-[(4-methylphenyl)sulfonyl]-1,2-dihydro-1′H-spiro[indole3,4′-piperidin]-1′-yl}nicotinamide (5). To a solution of B, t-butyl(2-{[6-chloropyridin-2-yl]carbonyl}amino)phenyl)carbamate (164 mg, 0.474mmol), in DMSO/PhMe (3 mL of a 2:1 solution) was added CC,1-[(4-methylphenyl)sulfonyl]-1,2-dihydrospiro[indole-3,4′-piperidine](487 mg, 1.42 mmol). After 12 h at 85° C. the reaction mixture wasdiluted with DMSO (5 mL) and purified by reverse-phase chromatography(25-100% MeCN/H₂O with 0.05% TFA). Formation oft-butyl(2-{[(6-{1-[(4-methylphenyl)sulfonyl]-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl}pyridin-3-yl)carbonyl]amino}phenyl)carbamate,the desired Boc-protected fused spiroamino nicotinamide, was confirmedby MS (ESI+): cal'd [M+H]⁺ 654.3, obs'd 654.2.

To a solution oft-butyl(2-{[(6-{1-[(4-methylphenyl)sulfonyl]-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl}pyridin-3-yl)carbonyl]amino}phenyl)carbamatein CH₂Cl₂ (4 mL) was added TFA (2 mL) dropwise. After 1 h at RT, thereaction mixture was concentrated and purified by reverse-phasechromatography (15-75% MeCN/H₂O with 0.05% TFA) to give 97 mg (37%, 2steps) ofN-(2-aminophenyl)-6-{1-[(4-methylphenyl)sulfonyl]-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl}nicotinamideafter neutralization of the TFA salt (sat.'d aq. NaHCO₃) confirmed by MS(ESI+): cal'd [M+H]⁺ 554.2, obs'd 554.2.

Example 2 SYNTHESIS OF ETHYL[1′-(5-{[(4-AMINOBIPHENYL-3-YL)AMINO]CARBONYL}PYRIDIN-2-YL)-2,3-DIHYDROSPIRO[INDENE-1,4′-PIPERIDIN]-3-YL]CARBAMATE

Ethyl[1′-(5-{[(4-Aminobiphenyl-3-yl)amino]carbonyl}pyridin-2-yl)-2,3-dihydrospiro[indene-1,4′-piperidin]-3-yl]carbamate (13). To a solution of D,t-butyl(3-{[(6-chloropyridin-3-yl)carbonyl]amino}biphenyl-4-yl)carbamate (75mg, 0.177 mmol), in DMSO (1 mL) was added i-PrNEt₂ (200 μL) and LL,ethyl 2,3-dihydrospiro[indene-1,4′-piperidin]-3-ylcarbamate (122 mg,0.442 mmol). After 8 h at 85° C. the reaction mixture was diluted withDMSO (5 mL) and purified by flash chromatography (25-100%EtOAc/hexanes). Formation of ethyl(1′-{5-[({4-[(t-butoxycarbonyl)amino]biphenyl-3-yl}amino)carbonyl]pyridin-2-yl}-2,3-dihydro-1′H-spiro[indene-1,4′-piperidin]-3-yl)carbamate,the desired Boc-protected fused spiroamino biphenyl nicotinamide, wasconfirmed by MS (ESI+): cal'd [M+H]⁺ 662.3, obs'd 662.3.

To a solution of ethyl(1′-{5-[({4-[(t-butoxycarbonyl)amino]biphenyl-3-yl}amino)carbonyl]pyridin-2-yl}-2,3-dihydro-1′H-spiro[indene-1,4′-piperidin]-3-yl)carbamatein CH₂Cl₂ (4 mL) was added TFA (2 mL) dropwise. After 1 h at RT, thereaction mixture was concentrated and purified by reverse-phasechromatography (25-100% MeCN/H₂O with 0.05% TFA) to give 109 mg (44%, 2steps) ofethyl[1′-(5-{[(4-aminobiphenyl-3-yl)amino]carbonyl}pyridin-2-yl)-2,3-dihydrospiro[indene-1,4′-piperidin]-3-yl]carbamate after neutralization of theTFA salt (sat.'d aq. NaHCO₃) confirmed by MS (ESI+): cal'd [M+H]⁺ 562.3,obs'd 562.3.

Example 3 SYNTHESIS OFN-[2-AMINO-5-(2-THIENYL)PHENYL]-6-(1,2-DIHYDRO-1′H-SPIRO[INDOLE-3,4′-PIPERIDN]-1′YL)NICOTINAMIDE

N-[2-amino-5-(2-thienyl)phenyl]-6-(1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′yl)nicotinamide(18). To a solution of F,tert-butyl[2-{[(6-chloropyridin-3-yl)carbonyl]amino}-4-(2-thienyl)phenyl]carbamate(228 mg, 0.530 mmol), in DMSO (4 mL) was added NEt₃ (222 μL) and AA,1,2-dihydrospiro[indole-3,4′-piperidine (300 mg, 1.59 mmol). After 8 hat 85° C. the reaction mixture was cooled to room temperature and pouredinto EtOAc and washed with saturated NaHCO₃, dried (MgSO₄) andconcentrated, giving the crude solid residue. The reaction mixture waspurified by flash chromatography (0-100% EtOAc/CH₂Cl₂). Formation oftert-butyl[2-({[6-(1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′yl)pyridine-3-yl]carbonyl}amino)-4-(2-thienyl)phenyl]carbamate,the desired Boc-protected fused spiroamino 2-thiophene biarylnicotinamide, was confirmed by MS (ESI+): cal'd [M+H]⁺ 582.2, obs'd582.2.

To a solution oftert-butyl[2-({[6-(1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′yl)pyridine-3-yl]carbonyl}amino)-4-(2-thienyl)phenyl]carbamatein CH₂Cl₂ (4 mL) was added TFA (4 mL) dropwise. After 1 h at RT, thereaction mixture was concentrated and the TFA salt was neutralized (2NNaOH) confirmed by MS (ESI+): cal'd [M+H]⁺ 482.2, obs'd 482.1.

Additional analogues were prepared using procedures similar to thosedescribed in the above examples. The compounds are all in neutral form.

CMPD # STRUCTURE COMPOUND NAME MS DATA 1

N-(2-aminophenyl)-6-(1,2- dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl)nicotinamide Cal'd [M⁺ + 1] 400.2, Obs'd 400.3 2

6-(1-acetyl-1,2-dihydro-1′H- spiro[indole-3,4′-piperidin]-1′-yl)-N-(2-aminophenyl)nicotinamide Cal'd [M⁺ + 1] 442.2, Obs'd 442.2 3

6-(1-acetyl-1,2-dihydro-1′H- spiro[indole-3,4′-piperidin]-1′-yl)-N-[2-amino-5-(2-thienyl)phenyl] nicotinamide Cal'd [M⁺ + 1] 524.2,Obs'd 524.2 4

6-(1-acetyl-1,2-dihydro-1′H- spiro[indole-3,4′-piperidin]-1′-yl)-N-[2-amino-5-(3-thienyl)phenyl] nicotinamide Cal'd [M⁺ + 1] 524.2,Obs'd 524.2 5

N-(2-aminophenyl)-6-{1-[(4- methylphenyl)sulfonyl]-1,2-dihydro-1′H-spiro[indole-3,4′- piperidin]-1′-yl}nicotinamide Cal'd [M⁺ +1] 554.2, Obs'd 554.2 6

N-(4-aminobiphenyl-3-yl)-6- (2,3-dihydro-1H,1′H-spiro[isoquinoline-4,4′- piperidin]-1′-yl)nicotinamide Cal'd [M⁺ + 1]490.3, Obs'd 490.3 7

N-(2-aminophenyl)-6-(1′H- spiro[indeno[2,1-b]pyridine-9,4′-piperidin]-1′-yl)nicotinamide Cal'd [M⁺ + 1] 448.2, Obs'd 448.2 8

N-(2-aminophenyl)-6-(1,3- diethyl-4,5-dihydro-1H,1′H-spiro[cyclopenta[c]pyrazole-6,4′- piperidin]-1′-yl)nicotinamide Cal'd[M⁺ + 1] 445.3, Obs'd 445.3 9

N-(2-aminophenyl)-6-(3-oxo- 2,3-dihydro-1′H-spiro[isoindole-1,4′-pipendin]-1′-yl)nicotinamide Cal'd [M⁺ + 1] 414.2, Obs'd 414.2 10

(3R)-1′-(5-{[(2-aminophenyl) amino]carbonyl}pyridin-2-yl)-N-methyl-2,3-dihydrospiro[indene-1,4′- piperidine]-3-carboxamide Cal'd[M⁺ + 1] 456.2, Obs'd 456.2 11

N-[2-amino-5-(3- thienyl)phenyl]-6-(4-oxo-3,4-dihydro-1′H-spiro[chromene- 2,4′-piperidin]-1′-yl)nicotinamide Cal'd[M⁺ + 1] 511.2, Obs'd 511.2 12

N-(4-aminobiphenyl-3-yl)-6-(1- oxo-1,4-dihydro-1′H-spiro[isochromene-3,4′- piperidin]-1′-yl)nicotinamide Cal'd [M⁺ + 1]505.2, Obs'd 505.2 13

ethyl [1′-(5-{[(4-aminobiphenyl- 3-yl)amino]carbonyl}pyridin-2-yl)-2,3-dihydrospiro[indene-1,4′- piperidin]-3-yl]carbamate Cal'd [M⁺ +1] 562.3, Obs'd 562.3 14

N-4-aminobiphenyl-3-yl)-6-(6- methoxy-1-oxo-4-phenyl-1,4-dihydro-1H′-spiro[isochromene- 3,4′-piperidin]-1′-yl)nicotinamide Cal'd[M⁺ + 1] 611.3, Obs'd 611.3 15

N-(2-aminophenyl)-6-(1-methyl- 2-oxo-5-phenyl-1,2-dihydro-1′H-spiro[1,4-benzodiazepine-3,4′- piperidin]-1′-yl)nicotinamide Cal'd [M⁺ +1] 531.2, Obs'd 531.2 16

N-(2-aminophenyl)-6-(1- isopropyl-6-methoxy-5-methyl-2-oxo-1,2-dihydro-1′H-spiro[indole- 3,4′-piperidin]-1′-yl)nicotinamideCal'd [M⁺ + 1] 500.3, Obs'd 500.3 17

N-(2-aminophenyl)-6-[5- (benzylamino)-1-isopropyl-2-oxo-1,2-dihydro-1′H-spiro[indole- 3,4′-piperidin]-1′-yl]nicotinamideCal'd [M⁺ + 1] 561.3, Obs'd 561.3 18

N-[2-amino-5-(2- thienyl)phenyl]-6-(1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]- 1′-yl)nicotinamide Cal'd [M⁺ + 1]482.2, Obs'd 482.2 19

N-[2-amino-5-(2- thienyl)phenyl]-6-(1′H-spiro[1-benzofuran-3,4′-piperidin]-1′- yl)nicotinamide Cal'd [M⁺ + 1] 483.2,Obs'd 483.2 20

N-[2-amino-5-(2-thienyl)phenyl]- 6-[1-(methylsulfonyl)-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]- 1′-yl]nicotinamide Cal'd [M⁺ + 1]560.2, Obs'd 560.2

Example 4 SYNTHESIS OF TERT-BUTYL1′-(5-{[(2-AMINOPHENYL)AMINO]CARBONYL}PYRIDIN-2-YL)-1H-SPIRO[ISOQUINOL-INE-4,4′-PIPERIDINE]-2(3H)—CARBOXYLATEANDN-(2-AMINOPHENYL)-6-(2,3-DIHYDRO-1H,1H-SPIRO[ISOQUINOLINE-4,4′-PIPERIDIN]-1′-YL)NICOTINAMIDE

tert-Butyl1′-(5-{[(2-Aminophenyl)amino]carbonyl}pyridin-2-yl)-1H-spiro[isoquinoline-4,4′-piperidine]-2(3H)-carboxylate(21). A mixture of methyl 6-chloronicotinoate (120 mg, 0.70 mmol),spirocycle DD (200 mg, 0.590 mmol) and NEt₃ (0.200 mL) in 2 mL of DMSOwas heated to 90° C. for 2 days. When cool, the mixture was poured intoEtOAc and washed with saturated NaHCO₃, dried (Na₂SO₄) and concentrated,giving the crude oily residue.

A solution of the methyl ester (65 mg, 0.15 mmol) in 2 mL of 1:1 H₂O/THFwas treated with LiOH.H₂O (25 mg, 0.60 mmol) and stirred for 12 hours,then concentrated to dryness. The residue was dissolved in 2 mL of DMF,treated with HOBt (50 mg, 0.37 mmol), EDC (100 mg, 0.52 mmol), diamine(50 mg, 0.46 mmol) and stirred for 12 hours. Concentrated to dryness.The residue was purified by reverse phase HPLC (20 to 100% MeCN/waterwith 0.05% TFA), then partitioned between EtOAc and saturated NaHCO₃.The organic layer was dried (Na₂SO₄) and concentrated giving 21; MS(ESI+): cal'd [M+H]⁺ 514.3, obs'd 514.3.

N-(2-Aminophenyl)-6-(2,3-dihydro-1H,1′H-spiro[isoquinoline-4,4′-piperidin]-1′-yl)nicotinamide(22). A mixture of 21 (4 mg, 0.008 mmol) in 1 mL of 1:1 DCM/TFA wasstirred for 3 h and concentrated to dryness giving the tris-TFA salt of22: MS (ESI+): cal'd [M+H]⁺ 414.2, obs'd 414.2.

Example 5 SYNTHESIS OFN-(4-AMINOBIPHENYL-3-YL)-4-(1H-SPIRO[ISOQUINOLINE-4,4′-PIPERIDIN]-2(3H-YLMETHYL)BENZAMIDE

N-(4-Aminobiphenyl-3-yl)-4-(1H-spiro[isoquinoline-4,4′-piperidin]-2(3H-ylmethyl)benzamide(23). The FDMP stratospheres resin (loading 1.5 mmol/g) (67 mg, 0.10mmol), 137 mg (0.5 mmol) of tert-butyl (3-aminobiphenyl-4-yl)carbamate(intermediate C), and 1 ml of 5% AcOH in DCE was added to ascintillation vial and allowed to shake overnight at room temperature.106 mg (0.5 mmol) of NaBH(OAc)₃ was added to the vial in 1 ml of 5% AcOHin DCE. The vial was capped and vented, and allowed to react for 3 daysat room temperature. The resin was washed with each of the followingsolvents 3× each and dried in vacuo: DMF, MeOH, H₂O, MeOH, and DCM.

The resin from the previous step (0.1 mmol) was added to a scintillationvial along with 2 ml of DCM and 51 mg (0.4 mmol) of DIEA. The vial wasshaken for 1 minute and 38 mg (0.2 mmol) of 4-chloromethyl benzoylchloride was added. The vial was capped and vented, and allowed to reactovernight at room temperature. The resin was washed with each of thefollowing solvents 3× each and dried in vacuo: DCM, DMF, H₂O, MeOH, andDCM.

The resin from the previous step (0.1 mmol) was added to a scintillationvial along with 214 mg (11.0 mmol) of proton sponge, 45 mg (0.3 mmol) ofNaI, 77 mg (0.5 mmol) of tert-butyl2,3-dihydro-1H,1′H-spiro[isoquinoline-4,4′-piperidine]-1′-carboxylate,and 2 ml of DMF. The resin was washed with each of the followingsolvents three times each and dried in vacuo: DMF, H₂O, MeOH, and DCM.

The resin from the previous step (0.1 mmol) was cleaved with 3 ml of 1:1DCM:TFA for 2 hours at room temperature. The filtrate was collected andpurified by HPLC to yield the product as a white solid: MS (ESI+): cal'd[M+H]⁺ 503.3, obs'd 503.3.

Example 6 SYNTHESIS OFN-(4-AMINOBIPHENYL-3-YL)-4-(2,3-DIHYDRO-1H,1′H-SPIRO[ISOQUINOLINE-4,4′-PIPERIDIN]-1′-YLCARBONYL)BENZAMIDE

N-(4-Aminobiphenyl-3-yl)-4-(2,3-dihydro-1H,1′H-spiro[isoquinoline-4,4′-piperidin]-1′-ylcarbonyl)benzamide(24). To a solution of stirring terephthaloyl chloride (50 mg, 0.246mmol) in 3 mL dichloromethane was added tert-butyl(3-aminobiphenyl-4-yl)carbamate C (70 mg, 0.246 mmol) slowly over aperiod of 10 minutes, followed by the addition of diisopropylethylamine(48 μL, 0.246 mmol). The reaction mixture was allowed to stir for 30min. at room temperature. tert-Butyl1H-spiro[isoquinoline-4,4′-piperidine]-2(3H)-carboxylate DD (83 mg,0.246 mmol) was then added, followed by the addition ofdiisopropylethylamine (48 μL, 0.246 mmol). The reaction mixture wasallowed to stir for 1 hour at room temperature. The reaction mixturebecame cloudy. Argonaut MP-Carbonate scavenging resin (255 g, 0.738mmol) was then added and stirred overnight at room temperature. Themixture was then fully dissolved by adding 3 mL dimethylformamide,filtered from scavenging resin, and concentrated. Added dichloromethane(1 mL) and stirred to form suspension, then treated with trifluoroaceticacid (1 mL). The reaction mixture was concentrated after 2 hours ofstirring at room temperature and the crude residue was purified byreverse-phase chromatography (5-70-95% acetonitrile/water with 0.1%formic acid). The appropriate fractions were combined and lyophilized.MS (ESI+): cal'd [M+H]⁺ 517.3, obs'd 517.2.

Example 7 SYNTHESIS OFN-(4-{[(4-AMINOBIPHENYL-3-YL)AMINO]CARBONYL}PHENYL)-2,3-DIHYDRO-1H,1′H-SPIRO[ISOQUINOLINE-4,4′-PIPERIDINE]-1′-CARBOXAMIDE

N-(4-{[(4-Aminobiphenyl-3-yl)amino]carbonyl}phenyl)-2,3-dihydro-1H,1′H-spiro[isoquinoline-4,4′-piperidine]-1′-carboxamide(25). To a solution of stirring 4-isocyanatobenzoyl chloride (50 mg,0.275 mmol) in 3 mL dichloromethane was added tert-butyl1H-spiro[isoquinoline-4,4′-piperidine]-2(3H)-carboxylate DD (93 mg,0.275 mmol) slowly over a period of 10 minutes. The reaction mixture wasallowed to stir for 30 min. at room temperature. tert-Butyl(3-aminobiphenyl-4-yl)carbamate C (78 mg, 0.275 mmol) was then added,followed by the addition of diisopropylethylamine (48 μL, 0.275 mmol).The reaction mixture was allowed to stir for 1 hour at room temperature.Argonaut MP-Carbonate scavenging resin (285 mg, 0.825 mmol) was thenadded and stirred overnight at room temperature. The mixture was thenfiltered from scavenging resin and concentrated. Added dichloromethane(1 mL) and stirred, then treated with trifluoroacetic acid (1 mL). Thereaction mixture was concentrated after 2 hours of stirring at roomtemperature and the crude residue was purified by reverse-phasechromatography (5-70-95% acetonitrile/water with 0.1% formic acid). Theappropriate fractions were combined and lyophilized. MS (ESI+): cal'd[M+H]⁺ 532.3, obs'd 532.2.

Example 8 Hdac Inhibition by Novel Compounds Hdac1-Flag Assay

Novel compounds were tested for their ability to inhibit histonedeacetylase, subtype 1 (HDAC1) using an in vitro deacetylation assay.The enzyme source for this assay was an epitope-tagged human HDAC1complex immuno-purified from stably expressing mammalian cells. Thesubstrate consisted of a commercial product containing an acetylatedlysine side chain (BIOMOL Research Laboratories, Inc., Plymouth Meeting,Pa.). Upon deacetylation of the substrate by incubation with thepurified HDAC1 complex, a fluorophore is produced that is directlyproportional to the level of deacetylation. Using a substrateconcentration at the Km for the enzyme preparation, the deacetylationassay was performed in the presence of increasing concentrations ofnovel compounds to semi-quantitatively determine the concentration ofcompound required for 50% inhibition (IC50) of the deacetylationreaction. The compounds of the instant invention described in theExamples and Tables above exhibit histone deacetylase inhibitoryactivity at concentrations of less than about 1 μM.

Example 9 HDAC Inhibition in Cell Lines ATP Assay

The novel compounds of the present invention were tested for theirability to inhibit proliferation of the human cervical cancer (HeLa) andcolon carcinoma (HCT116) cells.

In this assay, also referred to as the Vialight Assay, cellular ATPlevels are measured as a means of quantifying cellular proliferation.This assay makes use of a bioluminescent method from Cambrex (ViaLightPLUS, cat. #LT07-121). In the presence of ATP, luciferase convertsluciferin to oxyluciferin and light. The amount of light produced(emission at 565 nM) is measured and correlates with a relative amountof proliferation. Human cervical cancer (HeLa) or colon carcinoma(HCT116) cells were incubated with vehicle or increasing concentrationsof compound for 48, 72 or 96 hours. Cell proliferation was quantified byadding the cell lysis reagent (provided in the Vialight assay kit)directly to culture wells, followed by addition of the ATP-monitoringreagent (containing luciferase/luciferin). The amount of light producedis then measured (emission at 565 nM). The quantity of light produced,as measured by 565 nM absorbance, is directly proportional to the numberof living cells in culture.

While this invention has been particularly shown and described withreferences to embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the meaning of the invention described.Rather, the scope of the invention is defined by the claims that follow.

What is claimed is:
 1. A compound represented by the followingstructural formula:

wherein X is

A, B, D, E, G, J, K, M and P are independently selected from CR¹ ₂,NR^(1a), C(O) and O; R is NH₂ or OH;

is phenyl or pyridyl;

is phenyl; each R¹ is independently 1) hydrogen, 2) C₁-C₆ alkyl, 3) (CR⁶₂)_(n)R¹⁰, 4) (CR⁶ ₂)_(n)C(O)R⁴, 5) (CR⁶ ₂)_(n)C(O)OR⁴, 6) (CR⁶₂)_(n)C(O)NR⁵ ₂, 7) (CR⁶ ₂)_(n)S(O)₂R⁴, 8) (CR⁶ ₂)_(n)OR⁴, 9) (CR⁶₂)_(n)NR⁵(SO₂)R⁴, 10) (CR⁶ ₂)_(n)NR⁵ ₂, 11) (CR⁶ ₂)_(n)NR⁵C(O)OR⁴, 12)halo, or 13) aryl; each R^(1a) is independently 1) hydrogen, 2) C₁-C₆alkyl, 3) (CR⁶ ₂)_(n)R¹⁰, 4) (CR⁶ ₂)_(n)C(O)R⁴, 5) (CR⁶ ₂)_(n)C(O)OR⁴,6) (CR⁶ ₂)_(n)C(O)NR⁵ ₂, or 7) (CR⁶ ₂)_(n)S(O)₂R⁴; L¹ is a bond R³ ishydrogen, unsubstituted or substituted aryl, unsubstituted orsubstituted heteroaryl; each R⁴ is independently 1) H, 2) C₁-C₆ alkyl,3) aryl, or 4) heterocyclyl, wherein alkyl, aryl or heterocyclyl isoptionally substituted with C₁-C₆ alkyl; each R⁵ is independently 1)hydrogen, 2) C₁-C₆ alkyl, or 3) aryl, which may be optionallysubstituted with 1 to 3 substituents selected from C₁-C₆ alkyl, aryl,heteroaryl or halo; each R⁶ is independently 1) hydrogen, 2) C₁-C₆alkyl, 3) aryl, 4) OR¹¹, 5) halo, or 6) NR¹¹; wherein the alkyl or arylmay be optionally substituted with 1 to 3 substituents selected from thegroup consisting of C₁-C₆ alkyl, aryl, heteroaryl and halo; each R¹⁰ isindependently 1) aryl, and 2) heterocyclyl, which may be optionallysubstituted; each R¹¹ is independently hydrogen, C₁-C₆ alkyl, or aryl,wherein said alkyl and aryl groups are optionally substituted; n isindependently selected from 0, 1, 2, 3 and 4; q is 1; or a stereoisomeror a pharmaceutically acceptable salt thereof.
 2. The compound accordingto claim 1 wherein R is NH₂, or a stereoisomer or a pharmaceuticallyacceptable salt thereof.
 3. The compound according to claim 2 wherein:

is pyridinyl;

is phenyl; or a stereoisomer or a pharmaceutically acceptable saltthereof.
 4. The compound of claim 1 selected fromN-(2-aminophenyl)-6-(1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl)nicotinamide;6-(1-acetyl-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl)-N-(2-aminophenyl)nicotinamide;6-(1-acetyl-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl)-N42-amino-5-(2-thienyl)phenyl]nicotinamide;6-(1-acetyl-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl)-N-[2-amino-5-(3-thienyl)phenyl]nicotinamide;N-(2-aminophenyl)-6-{1-[(4-methylphenyl)sulfonyl]-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl]nicotinamide;N-(4-aminobiphenyl-3-yl)-6-(2,3-dihydro-1H,1′H-spiro[isoquinoline-4,4′-piperidin]-1′-yl)nicotinamide;N-(2-aminophenyl)-6-(1′H-spiro[indeno[2,1-b]pyridine-9,4′-piperidin]-1′-yl)nicotinamide;N-(2-aminophenyl)-6-(1,3-diethyl-4,5-dihydro-1H,1′H-spiro[cyclopenta[c]pyrazole-6,4′-piperidin]-1′-yl)nicotinamide;N-(2-aminophenyl)-6-(3-oxo-2,3-dihydro-1′H-spiro[isoindole-1,4′-piperidin]-1′-yl)nicotinamide;(3R)-1′-(5-{[(2-aminophenyl)amino]carbonyl}pyridin-2-yl)-N-methyl-2,3-dihydrospiro[indene-1,4′-piperidine]-3-carboxamide;N-[2-amino-5-(3-thienyl)phenyl]-6-(4-oxo-3,4-dihydro-1′H-spiro[chromene-2,4′-piperidin]-1′-yl)nicotinamide;N-(4-aminobiphenyl-3-yl)-6-(1-oxo-1,4-dihydro-1′H-spiro[isochromene-3,4′-piperidin]-1′-yl)nicotinamide;ethyl[1′-(5-{[(4-aminobiphenyl-3-yl)amino]carbonyl}pyridin-2-yl)-2,3-dihydrospiro[indene-1,4′-piperidin]-3-yl]carbamate;N-4-aminobiphenyl-3-yl)-6-(6-methoxy-1-oxo-4-phenyl-1,4-dihydro-1H′-spiro[isochromene-3,4′-piperidin]-1′-yl)nicotinamide;N-(2-aminophenyl)-6-(1-methyl-2-oxo-5-phenyl-1,2-dihydro-1′H-spiro[1,4-benzodiazepine-3,4′-piperidin]-1′-yl)nicotinamide;N-(2-aminophenyl)-6-(1-isopropyl-6-methoxy-5-methyl-2-oxo-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl)nicotinamide;N-(2-aminophenyl)-6-[5-(benzylamino)-1-isopropyl-2-oxo-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl]nicotinamide;N-[2-amino-5-(2-thienyl)phenyl]-6-(1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl)nicotinamide;N-[2-amino-5-(2-thienyl)phenyl]-6-(l′H-spiro[1-benzofuran-3,4′-piperidin]-1′-yl)nicotinamide;N-[2-amino-5-(2-thienyl)phenyl]-6-[1-(methylsulfonyl)-1,2-dihydro-1′H-spiro[indole-3,4′-piperidin]-1′-yl]nicotinamide;tert-butyll′-(5-{[(2-aminophenyl)amino]carbonyl}pyridin-2-yl)-1H-spiro[isoquinoline-4,4′-piperidine]-2(3H)-carboxylate;N-(2-aminophenyl)-6-(2,3-dihydro-1H,1′H-spiro[isoquinoline-4,4′-piperidin]-1′-yl)nicotinamide;N-(4-aminobiphenyl-3-yl)-4-(1H-spiro[isoquinoline-4,4′-piperidin]-2(3H)-ylmethyl)benzamide;N-(4-aminobiphenyl-3-yl)-4-(2,3-dihydro-1H,1′H-spiro[isoquinoline-4,4′-piperidin]-1′-ylcarbonyl)benzamide;N-(4-{[(4-aminobiphenyl-3-yl)amino]carbonyl}phenyl)-2,3-dihydro-1H,1′H-spiro[isoquinoline-4,4′-piperidine]-1′-carboxamide;or a stereoisomer or a pharmaceutically acceptable salt thereof.
 5. Apharmaceutical composition comprising a pharmaceutically effectiveamount of the compound according to claim 1, and a pharmaceuticallyacceptable carrier.
 6. A method for the treatment of cervical cancer orcolon carcinoma in a mammal comprising the step of administering atherapeutically effective amount of the compound of claim 1 to themammal.